Reagent Guide

Applied Biosystems StepOne™

and StepOnePlus™
Real-Time PCR Systems
Applied Biosystems StepOne™ and
StepOnePlus™ Real-Time PCR
Systems

Reagent Guide
© Copyright 2008, 2010 Applied Biosystems. All rights reserved.
Information in this document is subject to change without notice. Applied Biosystems assumes no responsibility for any errors that may appear in this
document.
APPLIED BIOSYSTEMS DISCLAIMS ALL WARRANTIES WITH RESPECT TO THIS DOCUMENT, EXPRESSED OR IMPLIED, IN-
CLUDING BUT NOT LIMITED TO THOSE OF MERCHANTABILITY OR FITNESS FOR A PARTICULAR PURPOSE. IN NO EVENT SHALL
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BASIS FOR SPECIAL, INCIDENTAL, INDIRECT, PUNITIVE, MULTIPLE OR CONSEQUENTIAL DAMAGES IN CONNECTION WITH OR
ARISING FROM THIS DOCUMENT, INCLUDING BUT NOT LIMITED TO THE USE THEREOF.
For Research Use Only. Not for use in diagnostic procedures.
NOTICE TO PURCHASER: Label License
The StepOne™ and StepOnePlus™ Real-Time PCR Systems are covered by US patents and corresponding claims in their non-US counterparts, owned
by Applied Biosystems Inc. No right is conveyed expressly, by implication, or by estoppel under any other patent claim, such as claims to apparatus,
reagents, kits, or methods such as 5′ nuclease methods. Further information on purchasing licenses may be obtained by contacting the Director of
Licensing, Applied Biosystems, 850 Lincoln Centre Drive, Foster City, California 94404, USA.
NOTICE TO PURCHASER:
PLEASE REFER TO THE USER'S GUIDE OR PRODUCT INSERT OF THE REAGENTS NAMED HEREIN FOR LIMITED LABEL LICENSE OR
DISCLAIMER INFORMATION.
TRADEMARKS:
Applied Biosystems, AB (Design), MicroAmp, Primer Express, and VIC are registered trademarks, and FAM, JOE, MicroAmp, MultiScribe, NED,
ROX, StepOne, StepOnePlus, TAMRA, TET, and VeriFlex are trademarks of Applied Biosystems or its subsidiaries in the U.S. and/or certain other
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AmpErase, AmpliTaq Gold, and TaqMan are registered trademarks of Roche Molecular Systems, Inc.
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All other trademarks are the sole property of their respective owners.

Part Number 4379704 Rev. E

06/2010
Contents Front Cover

Preface
How to Use This Guide . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . vii
How to Obtain More Information . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ix
How to Obtain Support . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . xii

Chapter 1 Introduction
About the StepOne™ and StepOnePlus™ Systems . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1-2
Supported Consumables . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1-4
Preparing for Experiments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1-6
Select the Experiment Type . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1-7
Select the Reagent Type . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1-8
Select the Assay Type . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1-9

Chapter 2 Reagent Overview

Overview . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2-2
TaqMan® Reagents . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2-2
SYBR® Green Reagents . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2-4
Selecting the Appropriate Reagent Type . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2-6
Minimizing DNA Contaminants . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2-7

Chapter 3 Quantitation Experiments

Section 3.1: About Quantitation Experiments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3-3
Overview . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3-4
Select a Quantitation Method . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3-5
Select 1-Step or 2-Step RT-PCR . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3-8
Select Singleplex or Multiplex PCR . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3-10
Select the Reagent Type . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3-12
Select the Assay Type . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3-12

Section 3.2: Design Guidelines . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3-15

Inventoried/Made to Order Assays . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3-16
TaqMan® Gene Expression Assays . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3-16

Applied Biosystems StepOne™ and StepOnePlus™ Real-Time PCR Systems Reagent Guide iii
 Custom TaqMan® Gene Expression Assays . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3-18
Select the Master Mix . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3-19
Design the Experiment . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3-20
Custom Assays . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3-21
Design Primers and Probes Using Primer Express® Software . . . . . . . . . . . . . . . 3-21
Select the Reagents . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3-24
Use the Recommended Thermal Cycling Conditions . . . . . . . . . . . . . . . . . . . . . . 3-27
Optimize Primer Concentrations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3-30
Optimize the Probe Concentration . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3-33
For More Information . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3-35

Chapter 4 Genotyping Experiments

Section 4.1: About Genotyping Experiments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4-3
Overview . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4-4
Select the Assay Type . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4-6

Section 4.2: Design Guidelines . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4-9

Pre-Designed/Validated Assays . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4-10
TaqMan® SNP Genotyping Assays . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4-10
TaqMan® Drug Metabolism Genotyping Assays . . . . . . . . . . . . . . . . . . . . . . . . . . 4-11
Pre-Developed TaqMan® Assay Reagents for Allelic Discrimination . . . . . . . . . . 4-12
Select the Master Mix . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4-13
Design the Experiment . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4-14
Custom Assays . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4-14
Custom TaqMan® SNP Genotyping Assays . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4-14
Select the Master Mix . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4-16
Design the Experiment . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4-16

Chapter 5 Presence/Absence Experiments

Section 5.1: About Presence/Absence Experiments . . . . . . . . . . . . . . . . . . . . . . . . . 5-3
Overview . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5-4
Select the Assay Type . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5-6

Section 5.2: Design Guidelines . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5-9

Inventoried/Made to Order Assays . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5-10
TaqMan® Gene Expression Assays . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5-10
Custom TaqMan® Gene Expression Assays . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5-12
TaqMan® Exogenous Internal Positive Control Reagents . . . . . . . . . . . . . . . . . . . 5-13
Select the Master Mix . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5-15
Design the Experiment . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5-16
Custom Assays . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5-16

iv Applied Biosystems StepOne™ and StepOnePlus™ Real-Time PCR Systems Reagent Guide
Appendix A Formulas
Standard Deviation Calculation Using the Standard Curve Method . . . . . . . . . . . . . . . A-2
Standard Deviation Calculation Using the Comparative Method . . . . . . . . . . . . . . . . . A-5
Formula for Comparative CT (ΔΔCT) Experiments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A-7

Appendix B Primer Limiting in Multiplex PCR

Appendix C Assay Design Guidelines

Appendix D Reagent Part Numbers

Quantitation Experiments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . D-2
Inventoried/Made to Order Assays . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . D-2
Custom Assays . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . D-2
Genotyping Experiments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . D-5
Pre-Designed/Validated Assays . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . D-5
Custom Assays . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . D-5
Presence/Absence Experiments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . D-7
Inventoried/Made to Order Assays . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . D-7
Custom Assays . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . D-8

Bibliography

Glossary

Index

Applied Biosystems StepOne™ and StepOnePlus™ Real-Time PCR Systems Reagent Guide v
vi Applied Biosystems StepOne™ and StepOnePlus™ Real-Time PCR Systems Reagent Guide
Preface

How to Use This Guide

About the System The guides listed below are shipped with the Applied Biosystems StepOne™ and
Documentation StepOnePlus™ Real-Time PCR Systems (StepOne™ and StepOnePlus™ systems).

Guide Purpose and Audience PN

Applied Biosystems StepOne™ and Explains how to perform experiments on the StepOne and 4376786
StepOnePlus™ Real-Time PCR Systems StepOnePlus systems. Each Getting Started Guide
Getting Started Guide for Genotyping functions as both:
Experiments
• A tutorial, using example experiment data provided with
Applied Biosystems StepOne™ and the Applied Biosystems StepOne™ Real-Time PCR 4376787
StepOnePlus™ Real-Time PCR Systems Software (StepOne™ software).
Getting Started Guide for • A guide for your own experiments.
Presence/Absence Experiments Intended for laboratory staff and principal investigators who
perform experiments using the StepOne or StepOnePlus
Applied Biosystems StepOne™ and 4376785
system.
StepOnePlus™ Real-Time PCR Systems
Getting Started Guide for Relative
Standard Curve and Comparative CT
Experiments

Applied Biosystems StepOne™ and 4376784

StepOnePlus™ Real-Time PCR Systems
Getting Started Guide for Standard Curve
Experiments

Applied Biosystems StepOne™ and Explains how to install and maintain the StepOne and 4376782
StepOnePlus™ Real-Time PCR Systems StepOnePlus systems.
Installation, Networking, and Maintenance
Intended for laboratory staff responsible for the installation
Guide
and maintenance of the StepOne or StepOnePlus system
Applied Biosystems StepOne™ and 4376783
StepOnePlus™ Real-Time PCR Systems
Installation Quick Reference Card

Applied Biosystems StepOne™ and Provides information about the reagents you can use on the 4379704
StepOnePlus™ Real-Time PCR Systems StepOne and StepOnePlus systems, including:
Reagent Guide
• An introduction to TaqMan® and SYBR® Green reagents
• Descriptions and design guidelines for the following
experiment types:
– Quantitation experiments
– Genotyping experiments
– Presence/absence experiments
Intended for laboratory staff and principal investigators who
perform experiments using the StepOne or StepOnePlus
system.

Applied Biosystems StepOne™ and StepOnePlus™ Real-Time PCR Systems Reagent Guide vii
Preface

Guide Purpose and Audience PN

Applied Biosystems StepOne™ and Explains how to prepare your site to receive and install the 4376768
StepOnePlus™ Real-Time PCR Systems StepOne and StepOnePlus systems.
Site Preparation Guide
Intended for personnel who schedule, manage, and perform
the tasks required to prepare your site for installation of the
StepOne or StepOnePlus system.

Applied Biosystems StepOne™ Real-Time Explains how to use the StepOne software to: NA
PCR Software Help
• Set up, run, and analyze experiments using the StepOne
and StepOnePlus systems.
• Monitor networked StepOne and StepOnePlus
instruments.
• Calibrate StepOne and StepOnePlus instruments.
• Verify the performance of StepOne and StepOnePlus
instruments with an RNase P run.
Intended for:
• Laboratory staff and principal investigators who perform
experiments using the StepOne or StepOnePlus system.
• Laboratory staff responsible for the installation and
maintenance of the StepOne or StepOnePlus system.

Assumptions This guide assumes that you:

• Are familiar with the Microsoft Windows® XP operating system.
• Are familiar with the Internet and Internet browsers.
• Know how to handle DNA and/or RNA samples and prepare them for PCR.
• Understand data storage, file transfer, and copying and pasting.
• Have networking experience, if you plan to integrate the StepOne or
StepOnePlus system into your existing laboratory data flow.

Text Conventions This guide uses the following conventions:

• Bold text indicates user action. For example:
Type 0, then press Enter for each of the remaining fields.
• Italic text indicates new or important words and is also used for emphasis.
For example:
Before analyzing, always prepare fresh matrix.
• A right arrow symbol () separates successive commands you select from a
drop-down or shortcut menu. For example:
Select FileOpen.

User Attention Two user attention words appear in Applied Biosystems user documentation. Each
Words word implies a particular level of observation or action as described below:
Note: – Provides information that may be of interest or help but is not critical to the
use of the product.
IMPORTANT! – Provides information that is necessary for proper instrument
operation, accurate reagent kit use, or safe use of a chemical.

viii Applied Biosystems StepOne™ and StepOnePlus™ Real-Time PCR Systems Reagent Guide
 Preface

Examples of the user attention words appear below:

Note: The Calibrate function is also available in the Control Console.
IMPORTANT! To verify your client connection, you need a valid user ID.

How to Obtain More Information

Related Other StepOne and StepOnePlus System Documents
Documentation The documents listed in the table below are not shipped with the StepOne or
StepOnePlus instrument.

Document PN

Applied Biosystems StepOne™ and StepOnePlus™ Real-Time PCR 4376791

Systems Installation Performance Verification Protocol

Applied Biosystems StepOne™ and StepOnePlus™ Real-Time PCR 4376790

Systems Installation Qualification-Operation Qualification Protocol

Applied Biosystems StepOne™ and StepOnePlus™ Real-Time PCR 4376788

Systems Planned Maintenance Protocol

Documents Related to Genotyping Experiments

Document PN

Allelic Discrimination Pre-Developed TaqMan® Assay Reagents Quick 4312212

Reference Card

Custom TaqMan® Genomic Assays Protocol 4367671

Custom TaqMan® SNP Genotyping Assays Protocol 4334431

Ordering TaqMan® SNP Genotyping Assays Quick Reference Card 4374204

®
Performing a Custom TaqMan SNP Genotyping Assay for 96-Well 4371394
Plates Quick Reference Card

Performing a TaqMan® Drug Metabolism Genotyping Assay for 96-Well 4367636

Plates Quick Reference Card

Pre-Developed TaqMan® Assay Reagents Allelic Discrimination 4312214

Protocol

TaqMan® Drug Metabolism Genotyping Assays Protocol 4362038

TaqMan® SNP Genotyping Assays Protocol 4332856

Applied Biosystems StepOne™ and StepOnePlus™ Real-Time PCR Systems Reagent Guide ix
Preface

Documents Related to Presence/Absence Experiments

Document PN

DNA Isolation from Fresh and Frozen Blood, Tissue Culture Cells, and 4343586
Buccal Swabs Protocol

NucPrep® Chemistry: Isolation of Genomic DNA from Animal and Plant 4333959
Tissue Protocol

PrepMan® Ultra Sample Preparation Reagent Protocol 4318925

Documents Related to Relative Standard Curve and Comparative CT Experiments

Document PN

Amplification Efficiency of TaqMan® Gene Expression Assays 127AP05

Application Note

Applied Biosystems High-Capacity cDNA Reverse Transcription Kits 4375575

Protocol

Custom TaqMan® Gene Expression Assays Protocol 4334429

Primer Express® Software Version 3.0 Getting Started Guide 4362460

TaqMan® Gene Expression Assays Protocol 4333458

User Bulletin #2: Relative Quantitation of Gene Expression 4303859

Documents Related to Standard Curve Experiments

Document PN

Amplification Efficiency of TaqMan® Gene Expression Assays 127AP05

Application Note

Custom TaqMan® Gene Expression Assays Protocol 4334429

Primer Express® Software Version 3.0 Getting Started Guide 4362460

TaqMan® Gene Expression Assays Protocol 4333458

User Bulletin #2: Relative Quantitation of Gene Expression 4303859

x Applied Biosystems StepOne™ and StepOnePlus™ Real-Time PCR Systems Reagent Guide
 Preface

Documents Related to the Reagent Guide

Document PN

Applied Biosystems High-Capacity cDNA Reverse Transcription Kits 4375575

Protocol

Custom TaqMan® Gene Expression Assays Protocol 4334429

Custom TaqMan® Genomic Assays Protocol: Submission Guidelines 4367671

Custom TaqMan® SNP Genotyping Assays Protocol 4334431

Power SYBR® Green PCR Master Mix and RT-PCR Protocol 4367218

Pre-Developed TaqMan® Assay Reagents Allelic Discrimination 4312214

Protocol

Primer Express® Software Version 3.0 Getting Started Guide 4362460

SYBR® Green PCR and RT-PCR Reagents Protocol 4304965

SYBR® Green PCR Master Mix and RT-PCR Reagents Protocol 4310251

TaqMan® Drug Metabolism Genotyping Assays Protocol 4362038

TaqMan® Exogenous Internal Positive Control Reagents Protocol 4308335

TaqMan® Fast Universal PCR Master Mix (2✕) Protocol 4351891

TaqMan® Gene Expression Assays Protocol 4333458

TaqMan® Gene Expression Master Mix Protocol 4371135

TaqMan® Genotyping Master Mix Protocol 4371131

TaqMan® SNP Genotyping Assays Protocol 4332856

TaqMan® Universal PCR Master Mix Protocol 4304449

User Bulletin #2: Relative Quantitation of Gene Expression 4303859

Using TaqMan® Endogenous Control Assays to Select an Endogenous 127AP08

Control for Experimental Studies Application Note

Note: For more documentation, see “How to Obtain Support” on page xii.

Obtaining The StepOne Software Help describes how to use each feature of the user interface.
Information from Access the Help from within the software by doing one of the following:
the Software Help • Press F1.
• Click in the toolbar.
• Select HelpStepOne Software Help.
To find topics of interest in the Help:
• Review the table of contents.
• Search for a specific topic.
• Search an alphabetized index.

Applied Biosystems StepOne™ and StepOnePlus™ Real-Time PCR Systems Reagent Guide xi
Preface

Send Us Your Applied Biosystems welcomes your comments and suggestions for improving its
Comments user documents. You can e-mail your comments to:
[email protected]
IMPORTANT! The e-mail address above is only for submitting comments and
suggestions relating to documentation. To order documents, download PDF files, or
for help with a technical question, go to http://www.appliedbiosystems.com, then
click the link for Support. (See “How to Obtain Support” on page Front Matter-xii).

How to Obtain Support

For the latest services and support information for all locations, go to
http://www.appliedbiosystems.com, then click the link for Support.
At the Support page, you can:
• Search through frequently asked questions (FAQs)
• Submit a question directly to Technical Support
• Order Applied Biosystems user documents, MSDSs, certificates of analysis,
and other related documents
• Download PDF documents
• Obtain information about customer training
• Download software updates and patches
In addition, the Support page provides access to worldwide telephone and fax
numbers to contact Applied Biosystems Technical Support and Sales facilities.
IMPORTANT! When directed to do so by this guide, or when you need to schedule
maintenance for your StepOne™ or StepOnePlus™ instrument (such as annual
planned maintenance or temperature verification/calibration), contact the
Applied Biosystems Care Center. To obtain a phone number for or to send an email
to the center, go to http://www.appliedbiosystems.com/support/contact.

xii Applied Biosystems StepOne™ and StepOnePlus™ Real-Time PCR Systems Reagent Guide
Introduction 1 1
This chapter covers:
About the StepOne™ and StepOnePlus™ Systems . . . . . . . . . . . . . . . . . . . . . . . . . 1-2
Supported Consumables . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1-4
Preparing for Experiments. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1-6
Select the Experiment Type . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1-7
Select the Reagent Type . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1-8
Select the Assay Type . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1-9

Applied Biosystems StepOne™ and StepOnePlus™ Real-Time PCR Systems Reagent Guide 1-1
Chapter 1 Introduction

About the StepOne™ and StepOnePlus™ Systems

There are two models available for this Real-Time PCR System:

System Features

Applied Biosystems StepOne™ Real-Time • 48-well platform

PCR System (StepOne™ system) • Three-color system

Applied Biosystems StepOnePlus™ Real- • 96-well platform

Time PCR System (StepOnePlus™ system) • Four-color system
• VeriFlex™ sample blocks

The StepOne and StepOnePlus systems use fluorescent-based polymerase chain

reaction (PCR) reagents to provide:
• Quantitative detection of target nucleic acid sequences (targets) using real-time
analysis.
• Qualitative detection of targets using post-PCR (endpoint) analysis.
• Qualitative analysis of the PCR product (achieved by melt curve analysis that
occurs post-PCR).

About Data The StepOne and StepOnePlus systems collect raw fluorescence data at different
Collection points during a PCR, depending on the type of run that the instruments perform:

Run Type Data Collection Point

Real-time runs Standard curve The instrument collects data following each
extension step of the PCR.
Relative standard
curve

Comparative CT
(ΔΔCT)

Post-PCR Genotyping The instrument collects data:

(endpoint) runs
Presence/absence • Before the PCR (For presence/absence
experiments, data collection before the PCR
is optional, but recommended.)
• (Optional) During the PCR. The instrument can
collect data during the run (real-time);
collecting data during the run can be helpful
for troubleshooting.
• After the PCR

Regardless of the run type, a data collection point or read on the StepOne™ or
StepOnePlus™ instrument consists of three phases:

1. Excitation – The instrument illuminates all wells of the reaction plate within
the instrument, exciting the fluorophores in each reaction.

1-2 Applied Biosystems StepOne™ and StepOnePlus™ Real-Time PCR Systems Reagent Guide
 Chapter 1 Introduction

2. Emission – The instrument optics collect the residual fluorescence emitted from
the wells of the reaction plate. The resulting image collected by the device
consists only of light that corresponds to the range of emission wavelengths.

3. Collection – The instrument assembles a digital representation of the residual

fluorescence collected over a fixed time interval. The StepOne™ software stores
the raw fluorescent image for analysis.
After a run, the StepOne software uses calibration data (spatial, dye, and
background) to determine the location and intensity of the fluorescent signals in each
read, the dye associated with each fluorescent signal, and the significance of the
signal.

About the Filters The StepOne and StepOnePlus systems use the following filters:

StepOne system StepOnePlus system

Filter Dye Filter Dye

1 FAM™ dye 1 FAM™ dye

SYBR® Green dye SYBR® Green dye

2 JOE™ dye 2 JOE™ dye

VIC® dye VIC® dye

3 ROX™ dye 3 TAMRA™ dye

NED™ dye

4 ROX™ dye

About the The StepOnePlus instrument contains six independently thermally regulated
VeriFlex™ VeriFlex™ blocks to help you optimize your thermal cycling conditions. You can set a
Technology different temperature for one or more of the VeriFlex blocks, creating up to six
different zones for samples, or you can set the same temperature for each of the
VeriFlex blocks.

For More For more information about any of the topics discussed in this guide, access the Help
Information from within Applied Biosystems StepOne™ Real-Time PCR Software v2.0 by
pressing F1, clicking in the toolbar, or selecting HelpStepOne Software Help.

Applied Biosystems StepOne™ and StepOnePlus™ Real-Time PCR Systems Reagent Guide 1-3
Chapter 1 Introduction

Supported Consumables
StepOne System The StepOne system supports the consumables listed below. These consumables are
for use with both standard and Fast reagents/protocols.
IMPORTANT! Use only Fast consumables (reaction plates, tube strips, and tubes)
with the StepOne and StepOnePlus systems, even when performing an experiment
with standard reagents.
Consumable Part Number

• MicroAmp™ Fast Optical 48-Well Reaction Plate • 4375816

• MicroAmp™ 48-Well Optical Adhesive Film • 4375323 and
4375928

• MicroAmp™ Fast 8-Tube Strip • 4358293

• MicroAmp™ Optical 8-Cap Strip • 4323032

• MicroAmp® Fast Reaction Tube with Cap • 4358297

™
• MicroAmp Fast 48-Well Tray • 4375282
• MicroAmp™ 48-Well Base Adaptor • 4375284
• MicroAmp™ 96-Well Support Base • 4379590

D F
E
G
B B
A H
H

C C C

# Consumable
A MicroAmp™ Fast Optical 48-Well Reaction Plate
™
B MicroAmp Fast 48-Well Tray
C MicroAmp™ 96-Well Support Base
D MicroAmp™ Optical 8-Cap Strip
E MicroAmp™ Fast 8-Tube Strip
F MicroAmp® Fast Reaction Tube with Cap
G MicroAmp™ 48-Well Optical Adhesive Film
H MicroAmp™ 48-Well Base Adaptor

1-4 Applied Biosystems StepOne™ and StepOnePlus™ Real-Time PCR Systems Reagent Guide
 Chapter 1 Introduction

StepOnePlus The StepOnePlus system supports the consumables listed below. These consumables
System are for use with both standard and Fast reagents/protocols.
IMPORTANT! Use only Fast consumables (reaction plates, tube strips, and tubes)
with the StepOne and StepOnePlus systems, even when performing an experiment
with standard reagents.

Consumable Part Number

• MicroAmp™ Fast Optical 96-Well Reaction Plate with • 4346906 and

Barcode 4366932
• MicroAmp™ Optical Adhesive Film • 4360954 and
4311971

• MicroAmp™ Fast 8-Tube Strip • 4358293

• MicroAmp™ Optical 8-Cap Strip • 4323032

• MicroAmp® Fast Reaction Tube with Cap • 4358297

• MicroAmp™ 96-Well Tray for VeriFlex™ Blocks • 4379983

• MicroAmp™ 96-Well Support Base • 4379590

• MicroAmp™ Adhesive Film Applicator • 4333183

• MicroAmp™ Cap Installing Tool (Handle) • 4330015

G
E
F
A
B B

C C C

# Consumable
™
A MicroAmp Fast Optical 96-Well Reaction Plate
B MicroAmp™ 96-Well Tray for VeriFlex™ Blocks
C MicroAmp™ 96-Well Support Base
D MicroAmp™ Optical 8-Cap Strip
E MicroAmp™ Fast 8-Tube Strip
F MicroAmp® Fast Reaction Tube with Cap
G MicroAmp™ Optical Adhesive Film

Applied Biosystems StepOne™ and StepOnePlus™ Real-Time PCR Systems Reagent Guide 1-5
Chapter 1 Introduction

Preparing for Experiments

General Workflow Before performing experiments on the StepOne and StepOnePlus systems, prepare
for the experiment as follows:
1. Select an experiment type (page 1-7).
2. Select the reagent type (page 1-8).
3. Select the assay type (page 1-9).

Information in This chapter provides general information on the experiment types, reagent types,
This Guide and assay types you can use with the StepOne and StepOnePlus systems.
Subsequent chapters provide specific information:

Chapter Description

Chapter 2, Reagent Overview • Describes and compares the TaqMan® and

SYBR® Green reagents.
• Provides information on minimizing DNA
contamination.

Chapter 3, Quantitation • Explains how the experiment type works.

Experiments • Provides a specific workflow for the experiment
type.
Chapter 4, Genotyping
Experiments • Provides design guidelines for each assay type.

Chapter 5, Presence/Absence
Experiments

1-6 Applied Biosystems StepOne™ and StepOnePlus™ Real-Time PCR Systems Reagent Guide
 Chapter 1 Introduction

Select the Experiment Type

In this guide, the term experiment refers to the entire process of performing a run
using the StepOne or StepOnePlus system, including setup, run and analysis. You
can perform the following types of experiments on the StepOne and StepOnePlus
systems:
• Quantitation, including:
– Standard curve
– Relative standard curve
– Comparative CT (ΔΔCT)
• Genotyping
• Presence/absence
Note: You can also perform melt curve analysis on the StepOne and StepOnePlus
systems. For more information, access the Help from within the StepOne software by
clicking in the toolbar or pressing F1.

Endpoint vs. The three experiment types can be categorized as real-time or endpoint experiments,
Real-Time as described below.
Experiments
Category Properties Experiment Type

Real-time • The instrument monitors the progress of the Quantitation

PCR as it occurs. ‡
• Data are collected throughout the PCR
process.
• Reactions are characterized by the point in
time during cycling when amplification of a
target is first detected. §

Endpoint • Data are collected at the end of the PCR Genotyping

process.
• Reactions are characterized by the quantity Presence/absence
of the target accumulated at the end of PCR.§
• The datapoint is the normalized intensity of
the reporter dye, or Rn.
Note: Some endpoint experiments also include
pre-PCR datapoints. If so, the system calculates
the delta Rn (ΔRn) value per the following
formula:
ΔRn = Rn (post-PCR read) – Rn (pre-PCR read)
where Rn = normalized reporter

‡ Kwok and Higuchi, 1989.

§ Saiki et al., 1985.

Applied Biosystems StepOne™ and StepOnePlus™ Real-Time PCR Systems Reagent Guide 1-7
Chapter 1 Introduction

Select the Reagent Type

You can use the following reagent types (chemistries) on the StepOne and
StepOnePlus systems:
• TaqMan® reagents
• SYBR® Green reagents
• Other fluorescent-based reagents

TaqMan TaqMan reagents include Applied Biosystems TaqMan® assays (preformulated

Reagents mixes that contain probe and primer sets) and Applied Biosystems TaqMan® master
mixes. You can use TaqMan reagents for the following experiment types:
• Quantitation, including:
– Standard curve
– Relative standard curve
– Comparative CT (ΔΔCT)
• Genotyping
• Presence/absence
IMPORTANT! Applied Biosystems does not recommend the use of TAMRA™ dye
as a reporter or quencher with the StepOne system. TAMRA dye may be used as a
reporter or quencher with the StepOnePlus system.

SYBR Green SYBR Green reagents include primers and master mixes that contain SYBR® Green
Reagents dye. You can use SYBR Green reagents for quantitation experiments:
• Standard curve
• Relative standard curve
• Comparative CT (ΔΔCT)
Note: You cannot perform multiplex PCR using SYBR Green reagents. For more
information, see “Select Singleplex or Multiplex PCR” on page 3-10.

Other Reagents You can use other fluorescent-based reagents on the StepOne and StepOnePlus
systems, but note the following when using the StepOne software:
• You must design your experiment using Advanced Setup instead of the Design
Wizard.
• For Applied Biosystems TaqMan and SYBR Green reagents, the StepOne
software automatically calculates reaction volumes in the Reaction Setup
screen.

1-8 Applied Biosystems StepOne™ and StepOnePlus™ Real-Time PCR Systems Reagent Guide
 Chapter 1 Introduction

Select the Assay Type

In the StepOne software, you can select the following assay types for quantitation,
presence/absence, and genotyping experiments:

Experiment Type Assay Type See...

Quantitation ‡ • Inventoried/Made to Order below

• Custom
Presence/absence

Genotyping • Pre-Designed/Validated page 1-10

• Custom
• User-Designed

‡ Quantitation experiments include standard curve, relative standard curve, and

comparative CT experiments.

Quantitation and Inventoried/Made to Order Assays

Presence/ For quantitation or presence/absence experiments, select the Inventoried/Made to
Absence Order assay type in the StepOne software if you are using:
Experiments
• TaqMan® Gene Expression Assays, Inventoried – Predesigned FAM™ dye-
labeled TaqMan® MGB (minor groove binder) probe and primer sets that can be
purchased off-the-shelf. The assay mix is available in a single, preformulated
20✕ tube.
• TaqMan® Gene Expression Assays, Made to Order – Predesigned FAM dye-
labeled TaqMan MGB probe and primer sets that are manufactured at the time
of order. The assay mix is available in a single, preformulated 20✕ tube.
• Custom TaqMan® Gene Expression Assays – FAM dye-labeled TaqMan
MGB probe and primer sets that are designed, synthesized, and formulated by
the Custom TaqMan® Genomic Assays service based on sequence information
that you submit. The assay mix is available in a single, preformulated 20✕ or
60✕ tube.
Note: To select the assay type in the StepOne software, go to the Reaction Setup
screen in either the Design Wizard or Advanced Setup, then select
Inventoried/Made to Order from the Assay Type dropdown menu.

Custom Assays
For quantitation and presence/absence experiments, select the Custom assay type in
the StepOne software if you are designing your own assays (primers and probes)
with Primer Express® Software and TaqMan or SYBR Green reagents. When
designing your own assays, follow the Applied Biosystems Assay Design Guidelines
to optimize results.
IMPORTANT! Applied Biosystems does not recommend the use of TAMRA™ dye
as a reporter or quencher with the StepOne system. TAMRA dye may be used as a
reporter or quencher with the StepOnePlus system.
Note: To select the assay type in the StepOne software, go to the Reaction Setup
screen in either the Design Wizard or Advanced Setup, then select Custom from the
Assay Type dropdown menu.

Applied Biosystems StepOne™ and StepOnePlus™ Real-Time PCR Systems Reagent Guide 1-9
Chapter 1 Introduction

Genotyping Pre-Designed/Validated Assays

Experiments For genotyping experiments, the Pre-Designed/Validated assay type includes:
• TaqMan® SNP Genotyping Assays – Predesigned FAM™ dye- and VIC® dye-
labeled TaqMan® MGB (minor groove binder) probes and primer sets that are
available as TaqMan® Pre-Designed SNP Genotyping Assays. The TaqMan Pre-
Designed SNP Genotyping Assays are manufactured at the time of order (Made
to Order). The assay mix is available in a single, preformulated 20✕ tube.
• TaqMan® Drug Metabolism Genotyping Assays – Predesigned FAM dye- and
VIC dye-labeled TaqMan MGB probes and primer sets that can be purchased
off-the-shelf (Inventoried). The assay mix is available in a single, preformulated
20✕ tube.
• Pre-Developed TaqMan® Assay Reagents for Allelic Discrimination
(TaqMan® PDARs for AD) – Predesigned FAM dye- and VIC dye-labeled
TaqMan MGB probes and primer sets that can be purchased off-the-shelf
(Inventoried). The assay mix is available in a single, preformulated 10✕ tube.
Note: To select a SNP assay in the StepOne software, go to the SNP Assays screen
in the Design Wizard or to the Plate Setup screen in Advanced Setup. In the SNP
Assays screen or Plate Setup screen, select an assay from the library or create a new
assay.

Custom Assays
For genotyping experiments, the Custom assay type includes the Custom TaqMan®
SNP Genotyping Assays. The Custom TaqMan SNP Genotyping Assays are FAM
dye- and VIC dye-labeled TaqMan MGB probes and primer sets that are designed,
synthesized, and formulated by the Custom TaqMan® Genomic Assays service based
on sequence information that you submit. The assay mix is available in a single,
preformulated 40✕ or 80✕ tube.
Note: To select a SNP assay in the StepOne software, go to the SNP Assays screen
in the Design Wizard or to the Plate Setup screen in Advanced Setup. In the SNP
Assays screen or Plate Setup screen, you can select an assay from the library or
create a new assay.

User-Designed Assays
If you want to design your own primers and probes for SNP assays, refer to the
Primer Express® Software Version 3.0 Getting Started Guide.
IMPORTANT! Applied Biosystems does not recommend the use of TAMRA™ dye
as a reporter or quencher with the StepOne system. TAMRA dye may be used as a
reporter or quencher with the StepOnePlus system.

1-10 Applied Biosystems StepOne™ and StepOnePlus™ Real-Time PCR Systems Reagent Guide
Reagent Overview 2 2
This chapter covers:
Overview . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2-2
TaqMan® Reagents . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2-2
SYBR® Green Reagents . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2-4
Selecting the Appropriate Reagent Type . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2-6
Minimizing DNA Contaminants . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2-7

Applied Biosystems StepOne™ and StepOnePlus™ Real-Time PCR Systems Reagent Guide 2-1
Chapter 2 Reagent Overview

Overview
Applied Biosystems has developed two reagent types (chemistries) that can be used
to detect PCR products on the Applied Biosystems StepOne™ and StepOnePlus™
Real-Time PCR Systems:
• TaqMan® reagents (below)
• SYBR® Green reagents (page 2-4)

TaqMan® Reagents
Experiment TaqMan® reagents include Applied Biosystems TaqMan® assays (preformulated
Types mixes that contain probe and primer sets) and Applied Biosystems TaqMan® master
mixes. The assays are specific to the target of interest. The master mixes contain the
remaining components needed for the PCR reaction. You can use TaqMan reagents
for the following experiment types:
• Quantitation, including:
– Standard curve
– Relative standard curve
– Comparative CT (ΔΔCT)
• Genotyping
• Presence/absence
IMPORTANT! Applied Biosystems does not recommend the use of TAMRA™ dye
as a reporter or quencher with the StepOne system. TAMRA dye may be used as a
reporter or quencher with the StepOnePlus system.

Development of Initially, intercalator dyes were used to measure real-time PCR products. The
TaqMan primary disadvantage of this detection method is that it detects accumulation of both
Reagents specific and nonspecific PCR products.
Real-time systems for PCR were improved by the introduction of fluorogenic-
labeled probes that use the 5′ nuclease activity of Taq DNA polymerase. The
availability of these fluorogenic probes enabled the development of a real-time
method for detecting only specific amplification products.

How the TaqMan TaqMan reagents use a fluorogenic probe to detect a specific PCR product as it
Reagents Work accumulates during the PCR. Here is how it works:
Step 1 – An oligonucleotide probe is constructed with a fluorescent reporter dye
bound to the 5′ end and a quencher on the 3′ end.
Step 2 – While the probe is intact, the proximity of the quencher greatly reduces the
fluorescence emitted by the reporter dye by fluorescence resonance energy transfer
(FRET; Förster resonance) (Förster, V. T. 1948) through space.

2-2 Applied Biosystems StepOne™ and StepOnePlus™ Real-Time PCR Systems Reagent Guide
 Chapter 2 Reagent Overview

Step 3 – If the target is present, the probe anneals between primer sites and is cleaved
by the 5′ nuclease activity of Taq DNA polymerase during extension. Cleavage of
the probe:
• Separates the reporter dye from the quencher, increasing the reporter dye signal.
• Removes the probe from the target strand, allowing primer extension to
continue to the end of the template strand. Thus, inclusion of the probe does not
inhibit the overall PCR process.
Step 4 – More reporter dye molecules are cleaved from their respective probes with
each cycle, resulting in an increase in fluorescence intensity proportional to the
quantity of amplicon produced. The higher the starting copy number of the nucleic
acid target, the earlier a significant increase in fluorescence is observed.
Figure 2-1 illustrates this process.

POLYMERIZATION STRAND DISPLACEMENT CLEAVAGE POLYMERIZATION COMPLETED

R R Q
FORWARD R = REPORTER R 3'
PRIMER R PROBE Q Q = QUENCHER Q Q
5' 3' 5' 3' 5' 3' 5'
3' 5' 3' 5' 3' 5' 3' 5'

5' 3' 5' 3' 5' 3' 5' 3'

5' 5' 5' 5'
REVERSE PRIMER

Step 1: A reporter (R) and Step 2: When both labels Step 3: During each Step 4: Once separated
a quencher (Q) are are attached to the probe, extension cycle, Taq DNA from the quencher, the
attached to the 5′ and 3′ reporter dye emission is polymerase cleaves the reporter dye emits its
ends of a TaqMan® probe. quenched. reporter dye from the characteristic
probe. fluorescence.

Figure 2-1 How the TaqMan reagents work

TaqMan® Probes Applied Biosystems offers two types of TaqMan® probes:

• TaqMan® MGB (minor groove-binder) probes with non fluorescent quencher
(NFQ)
• TaqMan® probes with TAMRA™ dye as quencher
IMPORTANT! Applied Biosystems does not recommend the use of TAMRA dye as a
reporter or quencher with the StepOne system. TAMRA dye may be used as a
reporter or quencher with the StepOnePlus system.

Applied Biosystems StepOne™ and StepOnePlus™ Real-Time PCR Systems Reagent Guide 2-3
Chapter 2 Reagent Overview

Applied Biosystems offers the following TaqMan probes for use on the StepOne
system or the StepOnePlus system:

Probe Assays 5′ Label Dye 3′ Label Dye MGB? System

®
TaqMan MGB TaqMan® Gene FAM™ dye NFQ Yes StepOne and
probe Expression Assays StepOne Plus
systems
TaqMan® SNP FAM™ and
Genotyping Assays VIC® dyes
TaqMan® MGB Custom TaqMan® Gene FAM™ dye
probe Expression Assays
Custom TaqMan® SNP FAM™and VIC®
Genotyping Assays dyes
Custom TaqMan® Custom assays FAM™, TET™,
MGB Probe NED™, or VIC®
dye
Custom TaqMan® Custom assays FAM™, TET™ or TAMRA™ No StepOnePlus
Probe VIC® dye dye system

TaqMan MGB Probes Recommended

Applied Biosystems recommends the general use of TaqMan MGB probes,
especially when conventional TaqMan probes exceed 30 nucleotides. The
TaqMan MGB probes contain:
• A fluorescent reporter dye at the 5′ end – Generates a signal when cleaved by
the 5′ nuclease activity of Taq DNA polymerase.
• A nonfluorescent quencher (NFQ) at the 3′ end – Allows Real-Time PCR
Systems to measure the reporter dye contributions more precisely than probes
with TAMRA dye because the quencher does not fluoresce.
• A minor groove binder (MGB) at the 3′ end – Increases the melting
temperature (Tm) of probes without increasing probe length (Afonina et al.,
1997; Kutyavin et al., 1997), thereby allowing the design of shorter probes.
Consequently, the TaqMan MGB probes exhibit greater differences in Tm
values between matched and mismatched probes; greater differences in Tm
values provide accurate genotyping.

SYBR® Green Reagents

Experiment SYBR® Green reagents include primers and master mixes that contain SYBR®
Types Green dye. You can use SYBR Green reagents for quantitation experiments:
• Standard curve
• Relative standard curve
• Comparative CT (ΔΔCT)
Note: You cannot perform multiplex PCR using SYBR Green reagents. For more
information, see “Select Singleplex or Multiplex PCR” on page 3-10.

2-4 Applied Biosystems StepOne™ and StepOnePlus™ Real-Time PCR Systems Reagent Guide
 Chapter 2 Reagent Overview

Development of Small molecules that bind to double-stranded DNA can be divided into two classes:
SYBR Green those that intercalate DNA and those that bind the minor groove of DNA. Higuchi
Reagents (Higuchi et al., 1992) used the intercalator ethidium bromide for real-time detection
of PCR. Hoechst 33258 is an example of a minor groove-binding dye whose
fluorescence increases when bound to double-stranded DNA (Higuchi et al., 1993).
Regardless of the binding method, there are at least two requirements for a DNA
binding dye for real-time detection of PCR products:
• Increased fluorescence when bound to double-stranded DNA
• No inhibition of PCR
Applied Biosystems has developed conditions that permit the use of the SYBR®
Green I dye in PCR without PCR inhibition and with increased sensitivity of
detection compared with ethidium bromide.

How the SYBR The SYBR Green reagents use the SYBR Green I dye to detect PCR products by
Green Reagents binding to double-stranded DNA formed during the PCR. Here is how it works:
Work
Step 1 – When added to a sample, SYBR Green I dye immediately binds to all
double-stranded DNA.
Step 2 – During the PCR, AmpliTaq Gold® DNA Polymerase amplifies the target,
which creates the PCR product, or “amplicon.” The double-stranded DNA is
denatured to single-stranded molecules and the SYBR Green I dye is released.
Step 3 – The primers anneal to the single-stranded DNA and the Amplitaq Gold
DNA Polymerase amplifies the target, creating more double stranded DNA. As the
PCR progresses, more amplicon is created.
Step 4 – The SYBR Green I dye then binds to each new copy of double-stranded
DNA that is generated during each PCR cycle. Since the SYBR Green I dye binds to
all double-stranded DNA, the result is an increase in fluorescence intensity
proportional to the quantity of double-stranded PCR product produced.
Figures 2-2 below illustrates this process.

Step 1: Reaction setup

The SYBR® Green I dye
fluoresces when bound to
double-stranded DNA.

Step 2: Denaturation
When the DNA is denatured into
single-stranded DNA, the
SYBR ® Green I dye is released and
the fluorescence is drastically reduced.
FORWARD
PRIMER Step 3: Polymerization
During extension, primers
anneal and PCR product
is generated.
REVERSE
PRIMER

Step 4: Polymerization completed

SYBR® Green I dye binds to the
double-stranded product,
resulting in a net increase in
fluorescence detected by the
instrument.

Figure 2-2 How the SYBR Green reagents work

Applied Biosystems StepOne™ and StepOnePlus™ Real-Time PCR Systems Reagent Guide 2-5
Chapter 2 Reagent Overview

Selecting the Appropriate Reagent Type

The TaqMan and SYBR Green reagents can be used for the experiment types listed
below.

Experiment Type

Presence/
Quantitation ‡ Genotyping
Reagent Type Absence
(Chapter 3) (Chapter 4)
(Chapter 5)

SYBR® Green reagents Yes Not Not

recommended recommended

TaqMan® reagents Yes Yes Yes

‡ Includes standard curve, relative standard curve, and comparative CT experiments.

IMPORTANT! Applied Biosystems does not recommend the use of TAMRA™ dye
as a reporter or quencher with the StepOne system. TAMRA dye may be used as a
reporter or quencher with the StepOnePlus system.

Considerations Quantitation experiments can be performed with either TaqMan or SYBR Green
for Quantitation reagents. Consider the following when choosing between the two reagent types:
Experiments

Reagent Type Description Advantage Limitation

TaqMan® TaqMan reagents use a • Provides increased Requires synthesis of a unique

reagents fluorogenic probe to enable specificity with the addition fluorogenic probe.
detection of a specific PCR of a fluorogenic probe.
product as it accumulates • Provides multiplex
during PCR cycles. capability.
• Includes preformulated
assays, optimized to run
under universal thermal
cycling conditions.
• Can be used for either 1- or
2-step RT-PCR.

SYBR® Green SYBR Green reagents use • Is economical (no probe Binds nonspecifically to all
reagents SYBR® Green I dye, a double- needed). double-stranded DNA
stranded DNA binding dye, to • Allows for melt curve sequences. To avoid false
detect PCR products as they analysis to measure the Tm positive signals, check for
accumulate during PCR of all PCR products. nonspecific product formation
cycles. using melt curve or gel
• Can be used for either 1- or analysis.
2-step RT-PCR.

2-6 Applied Biosystems StepOne™ and StepOnePlus™ Real-Time PCR Systems Reagent Guide
 Chapter 2 Reagent Overview

Minimizing DNA Contaminants

The DNA amplification capability of the PCR process makes special laboratory
practices necessary when you perform experiments using TaqMan or SYBR Green
reagents. Potential contamination can be introduced by samples with high DNA
concentrations, either from the DNA template controls or from PCR carryover.
In addition, due to the nonspecific nature of the SYBR Green I dye, any double-
stranded DNA will be detected. When using SYBR Green reagents, check for
nonspecific product formation by using melt curve or gel analysis. Take care to avoid
contamination with target DNA. Gene expression assays that span exon-exon
junctions minimize the effect of gDNA (genomic DNA) contaminants.

Using UNG to AmpErase® uracil-N-glycosylase (UNG) is a 26-kDa recombinant enzyme encoded

Minimize by the Escherichia coli uracil-N-glycosylase gene. This gene has been inserted into
Reamplification an E. coli host to direct expression of the native form of the enzyme (Kwok and
of Carryover Higuchi, 1989).
Products
UNG acts on single- and double-stranded dU-containing DNA. It acts by
hydrolyzing uracil-glycosidic bonds at dU-containing DNA sites. The enzyme
causes the release of uracil, thereby creating an alkali-sensitive apyrimidic site in the
DNA. The enzyme has no activity on RNA or dT-containing DNA (Longo et al.,
1990).

TaqMan Assays
For TaqMan® assays, AmpErase® UNG treatment can prevent the reamplification of
carryover PCR products from previous PCR reactions. When dUTP replaces dTTP in
PCR amplification, AmpErase UNG treatment can remove up to 200,000 copies of
amplicon per 50-μL reaction.
Note: AmpErase UNG (also abbreviated as UDG for uracil-DNA-glycosylase) is
included in some Applied Biosystems TaqMan master mix formulations and can also
be purchased individually. When purchasing TaqMan master mixes, check the
product information to see if the master mix contains AmpErase UNG.

SYBR Green I Dye Assays

For SYBR Green I dye assays, AmpErase UNG treatment can prevent the
reamplification of carryover PCR products from previous PCR reactions. Although
Power SYBR® Green PCR Master Mix and SYBR® Green PCR Master Mix do not
contain AmpErase UNG, they contain dUTP and are thus compatible with
AmpErase UNG. If contamination from PCR carryover is suspected, use
AmpErase UNG to troubleshoot the problem.
Note: AmpErase UNG can be purchased individually or as part of the
SYBR® Green PCR Core Reagents kit.

Applied Biosystems StepOne™ and StepOnePlus™ Real-Time PCR Systems Reagent Guide 2-7
Chapter 2 Reagent Overview

General PCR Use the following precautions to minimize sample contamination and PCR product
Practices carryover:
• Wear a clean lab coat (not previously worn while handling amplified PCR
products or used during sample preparation) and clean gloves when preparing
samples for PCR amplification. Change gloves whenever you suspect that they
are contaminated.
• Maintain separate areas, dedicated equipment, and supplies for:
– Sample preparation.
– PCR setup. Never bring amplified PCR products into the PCR setup area.
– PCR amplification.
– Analysis of PCR products.
• Open and close all sample tubes carefully. Avoid splashing or spraying PCR
samples.
• Use positive-displacement or air-displacement pipettors with filter-plugged tips.
Change tips after each use.
• Keep reactions and components capped as much as possible.
• Clean lab benches and equipment periodically with 10% bleach solution or 70%
ethanol.

2-8 Applied Biosystems StepOne™ and StepOnePlus™ Real-Time PCR Systems Reagent Guide
Quantitation Experiments 3 3
This chapter covers:
Section 3.1 About Quantitation Experiments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3-3
Section 3.2 Design Guidelines . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3-15

Applied Biosystems StepOne™ and StepOnePlus™ Real-Time PCR Systems Reagent Guide 3-1
Chapter 3 Quantitation Experiments

3-2 Applied Biosystems StepOne™ and StepOnePlus™ Real-Time PCR Systems Reagent Guide
 Chapter 3 Quantitation Experiments

Section 3.1 About Quantitation Experiments

This section covers:

Overview . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3-4
Select a Quantitation Method . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3-5
Select 1-Step or 2-Step RT-PCR . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3-8
Select Singleplex or Multiplex PCR . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3-10
Select the Reagent Type . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3-12
Select the Assay Type . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3-12

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Chapter 3 Quantitation Experiments

Overview
What Is a A quantitation experiment is a real-time experiment that measures the quantity of a
Quantitation target nucleic acid sequence (target) during each amplification cycle of the
Experiment? polymerase chain reaction (PCR). The target can be DNA, cDNA, or RNA.
Three types of quantitation experiments are discussed in this guide:
• Standard curve (page 3-5)
• Relative standard curve (page 3-5)
• Comparative CT (ΔΔCT) (page 3-6)

How Quantitation In real-time quantitation experiments, the reactions are characterized by the point in
Experiments time during cycling when amplification of a PCR product achieves a fixed level of
Work fluorescence, rather than the final quantity of PCR product accumulated after a fixed
number of cycles. An amplification plot graphically displays the fluorescence
detected over the number of cycles that were performed.
In the initial cycles of PCR, there is no significant change in fluorescence signal.
This predefined range of PCR cycles is called the baseline. First, the software
generates a baseline-subtracted amplification plot by calculating a mathematical
trend of the normalized fluorescent reporter signal (Rn values corresponding to the
baseline cycles). Then, an algorithm searches for the point on the amplification plot
at which the baseline-corrected normalized fluorescent reporter signal (delta Rn
[ΔRn] value) crosses a set threshold. The cycle at which the ΔRn value crosses the
threshold is defined as the CT.

Workflow Before performing quantitation experiments on the Applied Biosystems StepOne and
StepOnePlus™ Real-Time PCR Systems, prepare for the experiment as follows:
1. Select a quantitation method (page 3-5).
2. Select 1- or 2-step RT-PCR (page 3-8).
3. Select singleplex or multiplex PCR reactions (page 3-10).
4. Select the reagent type (page 3-12).
5. Select the assay type (page 3-12).
6. Review the design guidelines for the assay type you selected (Section 3.2 on
page 3-15).

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Select a Quantitation Method

About Standard The standard curve method is used to determine the absolute target quantity in
Curve samples. With the standard curve method, the StepOne software measures
Experiments amplification of the target in samples and in a standard dilution series. Data from the
standard dilution series are used to generate the standard curve. Using the standard
curve, the software interpolates the absolute quantity of target in the samples.

Components
The following components are required when setting up PCR reactions for standard
curve experiments:
• Sample – The sample in which the quantity of the target is unknown.
• Standard – A sample that contains known standard quantities; used in
quantitation experiments to generate standard curves.
• Standard dilution series – A set of standards containing a range of known
quantities. The standard dilution series is prepared by serially diluting
standards.
• Replicates – The total number of identical reactions containing identical
samples, components, and volumes.
• Negative Controls – Wells that contain water or buffer instead of sample
template. No amplification of the target should occur in negative control wells.

About Relative The relative standard curve method is used to determine relative target quantity in
Standard Curve samples. With the relative standard curve method, the StepOne software measures
Experiments amplification of the target and of the endogenous control in samples, in a reference
sample, and in a standard dilution series. Measurements are normalized using the
endogenous control. Data from the standard dilution series are used to generate the
standard curve. Using the standard curve, the software interpolates target quantity in
the samples and in the reference sample. The software determines the relative
quantity of target in each sample by comparing target quantity in each sample to
target quantity in the reference sample.
Relative standard curve experiments are commonly used to:
• Compare expression levels of a gene in different tissues.
• Compare expression levels of a gene in a treated sample vs. an untreated
sample.
• Compare expression levels of wild-type alleles vs. mutated alleles.

Components
The following components are required when setting up PCR reactions for relative
standard curve experiments:
• Sample – The sample in which the quantity of the target is unknown.
• Reference sample – The sample used as the basis for relative quantitation
results. For example, in a study of drug effects on gene expression, an untreated
control would be an appropriate reference sample. Also called calibrator.

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Chapter 3 Quantitation Experiments

• Standard – A sample that contains known standard quantities; used in

quantitation experiments to generate standard curves.
• Standard dilution series – A set of standards containing a range of known
quantities. The standard dilution series is prepared by serially diluting
standards.
• Endogenous control – A target or gene that should be expressed at similar
levels in all samples you are testing. The endogenous control is used to
normalize fluorescence signals for the target you are quantifying. Housekeeping
genes can be used as endogenous controls.
• Replicates – The total number of identical reactions containing identical
samples, components, and volumes.
• Negative Controls – Wells that contain water or buffer instead of sample
template. No amplification of the target should occur in negative control wells.

About The comparative CT (ΔΔCT) method is used to determine the relative target quantity
Comparative CT in samples. With the comparative CT method, the StepOne software measures
Experiments amplification of the target and of the endogenous control in samples and in a
reference sample. Measurements are normalized using the endogenous control. The
software determines the relative quantity of target in each sample by comparing
normalized target quantity in each sample to normalized target quantity in the
reference sample.
Comparative CT experiments are commonly used to:
• Compare expression levels of a gene in different tissues.
• Compare expression levels of a gene in a treated sample vs. an untreated
sample.
• Compare expression levels of wild-type alleles vs. mutated alleles.

Components
The following components are required when setting up PCR reactions for
comparative CT experiments:
• Sample – The sample in which the quantity of the target is unknown.
• Reference sample – The sample used as the basis for relative quantitation
results. For example, in a study of drug effects on gene expression, an untreated
control would be an appropriate reference sample. Also called calibrator.
• Endogenous control – A target or gene that should be expressed at similar
levels in all samples you are testing. The endogenous control is used to
normalize fluorescence signals for the target you are quantifying. Housekeeping
genes can be used as endogenous controls.
• Replicates – The total number of identical reactions containing identical
samples, components, and volumes.
• Negative Controls – Wells that contain water or buffer instead of sample
template. No amplification of the target should occur in negative control wells.

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Comparison of Consider the following when choosing between standard curve, relative standard
Quantitation curve, and comparative CT experiments:
Methods

Experiment
Description Advantage Limitation
Type

Standard curve Uses a standard curve to Allows comparisons against Because a standard curve
determine the absolute known standard quantities. must be constructed for each
quantity of a target in a target, standard curve
sample. Typically used for experiments require more
quantifying viral load. reagents and more space in
the reaction plate.

Relative Uses a standard curve to Requires the least amount of Because a standard curve
standard curve determine the change in validation because the PCR must be constructed for each
expression of a target in a efficiencies of the target and target, relative standard curve
sample relative to the same endogenous control do not experiments require more
target in a reference sample. need to be equivalent. reagents and more space in
Best for assays that have the reaction plate.
suboptimal PCR efficiency.

Comparative Uses arithmetic formulas to • Relative levels of target in • Suboptimal (low PCR
CT (ΔΔCT) determine the change in samples can be determined efficiency) assays may
expression of a target in a without the use of a produce inaccurate results.
sample relative to the same standard curve, provided • Before you use the
target in a reference sample. that the PCR efficiencies of comparative CT method,
Best for high-throughput the target and endogenous Applied Biosystems
measurements of relative gene control are relatively recommends that you
expression of many genes in equivalent. determine that the PCR
many samples. • Reduced reagent usage. efficiencies for the target
• More space is available in assay and the endogenous
the reaction plate. control assay are
approximately equal.

For More For more information on quantitation methods, refer to User Bulletin #2: Relative
Information Quantitation of Gene Expression.

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Chapter 3 Quantitation Experiments

Select 1-Step or 2-Step RT-PCR

Reverse transcription-polymerase chain reaction (RT-PCR) is used to quantify RNA.
RT-PCR can be performed as a 1-step or 2-step procedure.

About 1-Step In 1-step RT-PCR, you perform reverse transcription and the PCR in a single buffer
RT-PCR system (Figure 3-1). The reaction proceeds without the addition of reagents between
the RT and PCR steps. 1-step RT-PCR offers the convenience of a single-tube
preparation for RT and PCR amplification. However, the carryover prevention
enzyme, AmpErase® uracil-N-glycosylase (UNG), cannot be used with 1-step RT-
PCR. In 1-step RT-PCR, the presence of UNG would destroy the cDNA as it is being
made. For information about UNG, see “Using UNG to Minimize Reamplification of
Carryover Products” on page 2-7.

Single
Tube

Figure 3-1 Schematic representation of 1-step RT-PCR

About 2-Step In 2-step RT-PCR, you perform two separate reactions: one for RT and one for PCR
RT-PCR (Figure 3-2). 2-step RT-PCR is useful when detecting multiple transcripts from a
single cDNA reaction, or when storing a portion of the cDNA for later use. When
you perform PCR using dUTP as one of the bases, you can use AmpErase® UNG
enzyme to prevent carryover contamination. For information about UNG, see “Using
UNG to Minimize Reamplification of Carryover Products” on page 2-7.

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Tube 1
Oligo d(T) or random hexamer

Tube 2 PCR Step

Figure 3-2 Schematic representation of 2-step RT-PCR

Primers Used for For 1-step RT-PCR, sequence-specific reverse primers can be used for cDNA
cDNA Synthesis synthesis.
For 2-step RT-PCR, the following primers can be used for cDNA synthesis:
• Oligo d(T)16
• Random primers
• Sequence-specific reverse primers
The choice of primers for reverse transcription is best made after experimentally
evaluating all three priming systems. For short RNA sequences containing no hairpin
loops, any of the three priming systems works equally well. For longer RNA
transcripts or sequences containing hairpin loops, consider the following guidelines:.

Primers Selection Guidelines

Oligo d(T)16 • Use to reverse transcribe only eukaryotic mRNAs and

retroviruses with poly-A tails
• Avoid long mRNA transcripts or amplicons greater than
2 kilobases upstream from the poly-A site

Random primers • Try first for use with long reverse transcripts or reverse
transcripts containing hairpin loops
• Use to transcribe all RNA (rRNA, mRNA, and tRNA)

Sequence-specific • Use to reverse transcribe RNA-containing complementary

reverse primers sequences only
• Use in 1-step RT-PCR

Comparison of
Method Primers for cDNA Synthesis Comments
RT-PCR Methods
1-step Sequence-specific reverse primer Requires single reaction mix
RT-PCR
AmpErase® UNG cannot be used

2-step Random hexamers cDNA can be stored for later use

RT-PCR
AmpErase® UNG can be used
Oligo d(T)16
Requires two reaction mixes
Sequence-specific reverse primers

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Chapter 3 Quantitation Experiments

Select Singleplex or Multiplex PCR

You can perform a PCR reaction using either:
• Singleplex PCR – In singleplex PCR a single primer set is present in the
reaction tube or well. Only one target or endogenous control can be amplified
per reaction.
or
• Multiplex PCR – In multiplex PCR, two or more primer sets are present in the
reaction tube or well. Each set amplifies a specific target or endogenous control.
Typically, a probe labeled with FAM™ dye detects the target and a probe
labeled with VIC® dye detects the endogenous control.
IMPORTANT! SYBR® Green reagents cannot be used for multiplex PCR.

IMPORTANT! Applied Biosystems does not recommend the use of TAMRA™

dye as a reporter or quencher with the StepOne system. TAMRA dye may be
used as a reporter or quencher with the StepOnePlus system.

Target Primer Set

Endogenous Control
Primer Set
cDNA
Singleplex PCR Multiplex PCR
GR2331

Figure 3-3 Singleplex vs. multiplex PCR

About Multiplex In order to perform multiplex PCR you must:

PCR • Ensure that the endogenous control you have selected is more abundant (lower
CT value) than all of the targets that you are trying to quantify under all
conditions.
• Run the endogenous control assay as a primer-limited assay. The endogenous
control assay (for the more abundant template) in each reaction must be primer-
limited to avoid competitive PCR that may alter the CT of the less abundant
template.

Primer Limiting in Multiplex PCR

To generate an accurate multiplex assay, it is important that the amplification of one
species does not dominate amplification of the other species. Otherwise, the
amplification of a highly abundant species can prevent the less-abundant species
from amplifying efficiently.
If the less-abundant species does not amplify efficiently, your experiment may
produce inaccurate results or, in severe cases, detection of the less-abundant species
may be inhibited completely. You can avoid this situation by limiting the
concentrations of the primers used to amplify the more abundant species, thereby
“turning off ” the amplification soon after the CT has been established. However, a
primer-limited assay may be more susceptible to fluctuations in reaction conditions
than the primer non-limited target assay that it is normalizing. For more information,
see Appendix B, “Primer Limiting in Multiplex PCR.”

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Singleplex vs. Primer limiting in multiplex PCR becomes increasingly more complex as the number
Multiplex PCR of targets you quantify increases. When you analyze multiple numbers of targets, it
may be more effective to use singleplex PCR for the following reasons:
• In multiplex PCR, it may be difficult to find a suitable endogenous control, one
that:
– Is more abundant than all of the targets you are quantifying.
– Does not change expression levels with experimental conditions or across
different samples.
You would first have to run all of your target assays and endogenous control
assays in both the multiplex and singleplex formats, then compare CT values
from both formats to determine if there are any effects of the multiplexing on
your CT values.
• In singleplex PCR, any target that does not change expression levels with
experimental conditions or across samples may serve as an endogenous control.
Therefore, the more targets you have in a singleplex format, the higher the
probability that you will have one or more suitable endogenous controls against
which to normalize your remaining targets.
Consider the following when choosing between multiplex and singleplex PCR:

PCR Description Advantage Limitation

Singleplex A reaction in which a • No optimization is required for • Requires sample for both the
single target or TaqMan® assays. target and the endogenous
endogenous control is • Any target that does not change control.
amplified in the reaction expression levels with
tube or well. experimental conditions or across
samples may serve as an
endogenous control.
• Flexibility to use TaqMan® or
SYBR® Green reagents.

Multiplex A reaction in which • Reduces both the running costs • The endogenous control assay
more than one target or and the dependence on accurate must be run as a primer-limited
endogenous control is pipetting when splitting a sample assay.
amplified in the reaction into two separate tubes. • Requires validation and
tube or well. optimization.
• May not be as effective as
singleplex PCR when analyzing
multiple numbers of targets.
• You cannot use SYBR® Green
reagents.

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Chapter 3 Quantitation Experiments

Select the Reagent Type

TaqMan vs. You can perform quantitation experiments on the StepOne and StepOnePlus systems
SYBR Green with:
Reagents • TaqMan® reagents
• SYBR® Green reagents
For information on choosing between TaqMan and SYBR Green reagents, see
“Considerations for Quantitation Experiments” on page 2-6.

Select the Assay Type

When you design your experiments with the StepOne software, you can select the
following assay types for quantitation experiments:
• Inventoried/Made to Order (page 3-13)
• Custom (page 3-14)
This section lists the products available for each assay type.
Note: The assays are specific to the target of interest. The master mixes contain the
remaining components needed for the PCR reaction.

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Inventoried/Made
Product Attributes
to Order Assays
TaqMan® Gene • Predesigned, gene-specific primer and probe sets for
Expression Assays human, mouse, rat, Arabidopsis, Drosophila, C. elegans,
C. familiares (dog), and M. mulatta (Rhesus) genes.
• Probe is a FAM™ dye-labeled MGB probe.
• Provided in a convenient, single, 20✕ tube.
• Available as Inventoried or Made to Order assays.

TaqMan® Endogenous • Optimized, preformulated, ready-to-use endogenous

Control Assays control assays.
Note: FAM™ dye- • Cost-effective gene expression quantitation for human,
labeled TaqMan® mouse, rat, Arabidopsis, Drosophila, and any eukaryotic
Endogenous Control species.
Assays are included as • Choice of FAM™ dye or VIC® dye labels (primer-limited).
Inventoried TaqMan
Gene Expression Assays.

Custom TaqMan® Gene • Any species or organism.

Expression Assays • Target of your choice.
• Probe is a FAM™ dye-labeled MGB probe.
• Provided in a convenient, single, 205 tube.

TaqMan® Gene Tailored for precise quantitation by real-time PCR for routine
Expression Master Mix and challenging experiments:
• Sensitive detection down to 1 copy of target.
• Multiplex PCR for co-amplifying two targets in a single
reaction.
• Specificity for differentiation between gene family
members.
• Validated with TaqMan® Gene Expression Assays.
• Simplifies assay implementation by using one reagent for
all assays.

TaqMan® 2✕ Universal • Provides optimal performance for TaqMan® assays that

PCR Master Mix use cDNA or DNA as a template.
(with or without • Contains components that ensure excellent assay
AmpErase® UNG) performance.
• The use of one reagent for all assays simplifies the process
of assay implementation.

TaqMan® Fast Universal • Provides the same attributes listed above for TaqMan® 2✕
PCR Master Mix (2✕), No Universal PCR Master Mix.
AmpErase® UNG • Allows you to run quantitation experiments on the
StepOne™ and StepOnePlus™ systems in about 35 min.

IMPORTANT! Applied Biosystems does not recommend the use of TAMRA™ dye as
a reporter or quencher with the StepOne system. TAMRA dye may be used as a
reporter or quencher with the StepOnePlus system.
For guidelines on designing your experiments with Inventoried/Made to Order
assays, see page 3-16.

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Chapter 3 Quantitation Experiments

Custom Assays
Product Attributes

Custom TaqMan® Probes • Any species or organism.

and Primers • Choice of dye labels, quenchers, and synthesis scales.
• For use with the Primer Express® Software and Applied
Biosystems Assay Design Guidelines.

Primer Express® Software that designs primers and probes for real-time PCR.
Software

TaqMan® Gene Tailored for precise quantitation by real-time PCR for routine
Expression Master Mix and challenging experiments:
• Sensitive detection down to 1 copy of target.
• Multiplex PCR for co-amplifying two targets in a single
reaction.
• Specificity for differentiation between gene family
members.
• Validated with TaqMan® Gene Expression Assays.
• Simplifies assay implementation by using one reagent for
all assays.

TaqMan® 2✕ Universal • Provides optimal performance for TaqMan® assays that

PCR Master Mix use cDNA or DNA as a template.
(with or without • Contains components that ensure excellent assay
AmpErase® UNG) performance.
• The use of one reagent for all assays simplifies the process
of assay implementation.

TaqMan® Fast Universal • Provides the same attributes listed above for TaqMan® 2✕
PCR Master Mix (2✕), No Universal PCR Master Mix.
AmpErase® UNG • Allows you to run quantitation experiments on the
StepOne™ and StepOnePlus™ systems in about 35 min.

Power SYBR® Green • Highly sensitive quantitation enables low-copy-number

PCR Master Mix detection (as few as two copies of a target gene).
• Detects double-stranded DNA, so specific probes are not
required.
• Contains highly purified AmpliTaq Gold® DNA polymerase
LD to minimize nonspecific product formation (including
primer-dimer).
• For use with the Primer Express® Software and Applied
Biosystems Assay Design Guidelines.

SYBR® Green PCR • Detects double-stranded DNA, so specific probes are not
Master Mix required.
• For standard applications when high sensitivity is not
required.
• Contains AmpliTaq Gold® DNA polymerase to minimize
nonspecific product formation (including primer-dimer).
• For use with the Primer Express® Software and Applied
Biosystems Assay Design Guidelines.

IMPORTANT! Applied Biosystems does not recommend the use of TAMRA™ dye as
a reporter or quencher with the StepOne system. TAMRA dye may be used as a
reporter or quencher with the StepOnePlus system.
For guidelines on designing your experiments with Custom assays, see page 3-21.

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Section 3.2 Design Guidelines

This section covers:

Inventoried/Made to Order Assays . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3-16
TaqMan® Gene Expression Assays . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3-16
Custom TaqMan® Gene Expression Assays . . . . . . . . . . . . . . . . . . . . . . . . 3-18
Select the Master Mix . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3-19
Design the Experiment . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3-20
Custom Assays . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3-21
Design Primers and Probes Using Primer Express® Software . . . . . . . . . . 3-21
Select the Reagents . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3-24
Use the Recommended Thermal Cycling Conditions . . . . . . . . . . . . . . . . . 3-27
Optimize Primer Concentrations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3-30
Optimize the Probe Concentration . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3-33
For More Information . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3-35

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Chapter 3 Quantitation Experiments

Inventoried/Made to Order Assays

Workflow If you select the Inventoried/Made to Order assay type in the StepOne™ software,
Applied Biosystems recommends that you follow the workflow below:
1. Select the assay:
• TaqMan® Gene Expression Assays (below).
• Custom TaqMan® Gene Expression Assays (page 3-18).
2. Select the master mix (page 3-19).
3. Design the experiment using the StepOne software (page 3-20).

TaqMan® Gene Expression Assays

Product TaqMan® Gene Expression Assays are a comprehensive collection of Inventoried

Description and Made to Order probe and primer sets for performing quantitation experiments on
human, mouse, rat, Arabidopsis, Drosophila, C. elegans, C. familiares (dog), and
M. mulatta (Rhesus) genes.
The assays:
• Use TaqMan® reagents to amplify and detect the target in cDNA samples.
• Are designed using an automated design and quality-controlled pipeline.
Inventoried assays are manufactured and placed in inventory; Made to Order
assays are predesigned and manufactured when ordered.
• Are designed and optimized to work with an Applied Biosystems TaqMan®
master mix, using universal thermal cycling conditions.
• When possible, amplify target cDNA without amplifying genomic DNA
(m suffix in assay ID) by designing probes that cross exon-exon junctions.

Product All TaqMan Gene Expression Assays require:

Requirements • Three components:
– 1 to 100 ng of cDNA sample (converted from RNA) per well, with all wells
in a study having the same amount of cDNA.
– 20✕ Gene Expression Assay Mix (specific for each target). Each assay mix
consists of two unlabeled PCR primers and a FAM™ dye-labeled TaqMan®
MGB (minor groove binder) probe in a preformulated 20✕ mix. 1✕ final
concentrations are 250 nM for the probe and 900 nM for each primer.
– TaqMan® Gene Expression Master Mix, TaqMan® Universal PCR Master
Mix (with or without AmpErase® UNG), or TaqMan® Fast Universal PCR
Master Mix, No AmpErase® UNG.
• Only one PCR amplification step during each PCR cycle and a simultaneous
real-time reading to obtain results.

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Available Assays TaqMan Gene Expression Assays are available for human, mouse, rat, Arabidopsis,
Drosophila, C. elegans, C. familiares (dog), and M. mulatta (Rhesus) genes. The part
numbers are:
• PN 4331182 for Inventoried assays
• PN 4351372 for Made to Order assays
The prefix of the assay name indicates the species for which the assay was designed:
Hs for Homo sapiens (human), Mm for Mus musculus (mouse), Rn for Rattus
norvegicus (rat), At for Arabidopsis thaliana, Dm for Drosophila melanogaster,
Ce for C. elegans, Cf for C. familiares (dog), and Rh for M. mulatta (Rhesus).
The suffix of the assay name indicates the assay placement, as described in the table
below.

Suffix Description

_m The assay’s probe spans an exon junction; the assay does not detect
genomic DNA.

_s The assay’s primers and probes are designed within a single exon; the assay
does detect genomic DNA.

_g The assay may detect genomic DNA; the assay’s primers and probes may be
within a single exon.

_mH The assay was designed to a transcript belonging to a gene family with high
sequence homology. The assay provides between 10 CT and 15 CT difference
_sH between the target gene and the gene with the closest sequence homology.
Therefore, the assay detects the target transcript with 1000- to 30,000-fold
_gH greater discrimination (sensitivity) than the closest homologous transcript, if
they are present at the same copy number in a sample.

_u The assay’s amplicon spans an exon junction and the probe sits completely in
one of the spanned exons.

TaqMan® Endogenous Control Assays

TaqMan® Endogenous Control Assays are available as:
• Inventoried TaqMan Gene Expression Assays (PN 4331182) – Each assay
contains a FAM™ dye-labeled TaqMan® MGB probe in a single, preformulated
20✕ tube.
• Individual control assays for all human, mouse, and rat species (various part
numbers) – Each assay contains either a FAM™ dye-labeled TaqMan® MGB
probe, a VIC® dye-labeled TaqMan® MGB probe, or a TAMRA™ dye-labeled
probe. TaqMan Endogenous Controls with VIC dye labels are primer-limited.
IMPORTANT! Applied Biosystems does not recommend the use of TAMRA™ dye as
a reporter or quencher with the StepOne system. TAMRA dye may be used as a
reporter or quencher with the StepOnePlus system.

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For More • For information on the latest available products and specific product uses, refer
Information to the Applied Biosystems Web site:
http://www.appliedbiosystems.com/
a. On the Home page, under TaqMan® Products, select TaqMan® Gene
Expression Assays.
b. On the Gene Expression Assays & Arrays page:
• Under Individual Assays, select TaqMan® Gene Expression Assays.
This option links to all TaqMan Gene Expression Assays, including
TaqMan Endogenous Control Assays that contain FAM dye-labeled
probes.
or
• Under Individual Control Assays, select TaqMan® Endogenous
Control Assays. This option links to the individual TaqMan
Endogenous Control Assays (that contain FAM dye-labeled TaqMan
MGB probes, VIC dye-labeled TaqMan MGB probes, or TAMRA
dye-labeled probes).
• For information on Custom TaqMan Endogenous Control Assays, refer to the
Using TaqMan® Endogenous Control Assays to Select an Endogenous Control
for Experimental Studies Application Note.
• For information on preparing PCR reactions using the TaqMan Gene Expression
Assays, refer to the TaqMan® Gene Expression Assays Protocol.

Custom TaqMan® Gene Expression Assays

Product Custom TaqMan® Gene Expression Assays are TaqMan probe and primer sets that
Description are designed, synthesized, and formulated by the Custom TaqMan® Genomic Assays
service based on sequence information that you submit. Custom TaqMan Gene
Expression Assays allow you to perform quantitation experiments on any gene or
splice variant in any organism.
The assays:
• Use TaqMan® reagents to amplify and detect the target in cDNA samples.
• Are developed using proprietary assay-design software.
• Are designed and optimized to work with an Applied Biosystems TaqMan®
master mix, using universal thermal cycling conditions.

Product All Custom TaqMan Gene Expression Assays require:

Requirements • A submission file that includes your target sequence. You create the submission
file using free File Builder software, then submit the file to the Custom
TaqMan® Genomic Assays service.
• Three components:
– 1 to 100 ng of cDNA sample (converted from RNA) per well, with all wells
in a study having the same amount of cDNA.
– 20✕ Gene Expression Assay or 60✕ Gene Expression Assay (specific for
each target). Each assay consists of two target-specific primers and a FAM™
dye-labeled TaqMan MGB probe in a preformulated 20✕ or 60✕ mix. 1✕
final concentrations are 250 nM for the probe and 900 nM for each primer.

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 Chapter 3 Quantitation Experiments

– TaqMan® Gene Expression Master Mix, TaqMan® Universal PCR Master

Mix (with or without AmpErase® UNG), or TaqMan® Fast Universal PCR
Master Mix, No AmpErase® UNG.
• Only one PCR amplification step during each PCR cycle and a simultaneous
real-time reading to obtain results.

For More • For information on the latest available products and specific product uses, refer
Information to the Applied Biosystems Web site:
http://www.appliedbiosystems.com/
a. On the Home page, under TaqMan® Products, select TaqMan® Gene
Expression Assays.
b. On the Gene Expression Assays & Arrays page, under Individual Assays,
select Custom TaqMan® Gene Expression Assays.
• For information on ordering Custom TaqMan Gene Expression Assays, refer to
the Custom TaqMan® Genomic Assays Protocol: Submission Guidelines.
• For information on preparing PCR reactions using the Custom TaqMan Gene
Expression Assays, refer to the Custom TaqMan® Gene Expression Assays
Protocol.

Select the Master Mix

Available Master Applied Biosystems Inventoried/Made to Order assays for quantitation experiments
Mixes are designed to work with the following TaqMan® master mixes:

Master Mix Part Number

TaqMan® Gene Expression Master Mix, 1-Pack (1 × 5 mL), 4369016

200 reactions

TaqMan® Gene Expression Master Mix, 10-Pack (10 × 5 mL), 4369542

2000 reactions

TaqMan® Fast Universal PCR Master Mix (2✕), No AmpErase® UNG, 4352042
250 Reactions

TaqMan® 2✕ Universal PCR Master Mix, 200 Reactions 4304437

TaqMan® 2✕ Universal PCR Master Mix, 2000 Reactions 4326708

10-Pack, TaqMan® 2✕ Universal PCR Master Mix 4305719

TaqMan® 2✕ Universal PCR Master Mix, No AmpErase® UNG, 200 4324018

Reactions

TaqMan® 2✕ Universal PCR Master Mix, No AmpErase® UNG, 2000 4326614

Reactions

10-Pack, TaqMan® 2✕ Universal PCR Master Mix, No AmpErase® 4324020

UNG

Applied Biosystems StepOne™ and StepOnePlus™ Real-Time PCR Systems Reagent Guide 3-19
Chapter 3 Quantitation Experiments

For More For information on using the TaqMan reagents, refer to:
Information • TaqMan® Fast Universal PCR Master Mix (2✕) Protocol
• TaqMan® Gene Expression Master Mix Protocol
• TaqMan® Universal PCR Master Mix Protocol

Design the Experiment

Use the StepOne For Applied Biosystems Inventoried/Made to Order assays, use the StepOne
Software software to design your quantitation experiments. The StepOne software
automatically calculates volumes for the:
• Reaction mix components
• Sample dilutions
• (Standard curve and relative standard curve experiments only) Standard
dilution series
Note: To select the Inventoried/Made to Order assay type in the StepOne software,
go to the Reaction Setup screen in either the Design Wizard or Advanced Setup, then
select Inventoried/Made to Order from the Assay Type dropdown menu.

For More For information on designing and performing quantitation experiments on the
Information StepOne and StepOnePlus systems, refer to:
• Applied Biosystems StepOne™ and StepOnePlus™ Real-Time PCR Systems
Getting Started Guide for Standard Curve Experiments
• Applied Biosystems StepOne™ and StepOnePlus™ Real-Time PCR Systems
Getting Started Guide for Relative Standard Curve and Comparative CT
Experiments

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 Chapter 3 Quantitation Experiments

Custom Assays
Workflow If you select the Custom assay type in the StepOne™ software for a quantitation
experiment (that is, you are designing your own primers and probes), Applied
Biosystems recommends that you follow the workflow for the Applied Biosystems
Assay Design Guidelines:
1. Design primers and probes using Primer Express® Software (below).
2. Select the appropriate reagents (page 3-24).
IMPORTANT! Applied Biosystems does not recommend the use of TAMRA™
dye as a reporter or quencher with the StepOne system. TAMRA dye may be
used as a reporter or quencher with the StepOnePlus system.
3. Use the recommended thermal cycling conditions (page 3-27).
4. Begin with default primer and probe concentrations. If needed, optimize the
primer concentrations (page 3-30) and probe concentrations (page 3-33).
IMPORTANT! These steps provide a rapid and reliable system for assay design and
optimization only when used in their entirety. Adopt the system as a whole to
achieve the highest level of success. For a more detailed description of Applied
Biosystems Assay Design Guidelines, see Appendix C.
Note: To select the Custom assay type in the StepOne software, go to the Reaction
Setup screen in either the Design Wizard or Advanced Setup, then select Custom
from the Assay Type dropdown menu.

Design Primers and Probes Using Primer Express® Software

The Primer Express® Software uses recommended parameters to select primers and
probes based on the DNA sequence that you provide.
If you are designing your own assay, follow the summary of the primer and probe
design guidelines for quantitation experiments on page 3-23. For a detailed
discussion of these guidelines see “About the Primer and Probe Design Guidelines”
on page 3-22.
IMPORTANT! Applied Biosystems does not recommend the use of TAMRA™ dye
as a reporter or quencher with the StepOne system. TAMRA dye may be used as a
reporter or quencher with the StepOnePlus system.
Note: Even though a probe is not required for SYBR® Green I dye detection, it is
still a good idea to use Primer Express software to select a primer and probe set when
you design an assay for SYBR® Green reagents. Although no probe will be used, the
primers will meet all required criteria; if you need to convert the assay to TaqMan
reagents to obtain higher specificity, you can find the probe immediately in the
original Primer Express software document.

Selecting an Selecting a good amplicon site ensures amplification of the target mRNA/cDNA
Amplicon Site for without co-amplifying the genomic sequence, pseudogenes, and other related genes.
Quantitation SYBR Green reagents can be useful for screening amplicon sites for gene
Assays expression.

Applied Biosystems StepOne™ and StepOnePlus™ Real-Time PCR Systems Reagent Guide 3-21
Chapter 3 Quantitation Experiments

Guidelines
• The amplicon should span one or more introns to avoid amplification of the
target gene in genomic DNA.
• The primer pair should be specific to the target gene to avoid amplification of
pseudogenes or other related genes.
• When designing primers, use Primer Express software guidelines.
• If no good sequence is found, it may be necessary to examine the sequence and
redesign the amplicon or simply screen for more sites.
If the gene you are studying does not have introns, it is not possible to design an
amplicon that will amplify the mRNA sequence without amplifying the genomic
sequence. In this case, run a control of your RNA sample that has not been reverse-
transcribed (RT minus controls).

About the Primer Selection of Small Amplicons

and Probe Design An important default parameter in Primer Express software is the selection of
Guidelines amplicons in the 50- to 150-basepair range. Small amplicons are favored because
they promote high-efficiency amplification.
In addition, high-efficiency assays enable relative quantitation to be performed using
the comparative CT (ΔΔCT) method (Livak and Schmittgen, 2001). This method
increases sample throughput by eliminating the need for standard curves.

G/C Content
Whenever possible, select primers and probes in a region with a G/C content of
30 to 80%. Regions with a G/C content >80% may not denature well during thermal
cycling, leading to a less efficient reaction. G/C-rich sequences are susceptible to
nonspecific interactions that may reduce reaction efficiency and produce nonspecific
signal in assays using SYBR Green reagents. Avoid primer and probe sequences
containing runs of four or more G bases.

Melting Temperature
When you select primers and probes with the recommended melting temperature
(Tm), you can use universal thermal cycling conditions. Applied Biosystems
recommends that the probe Tm be 10 °C higher than that of the primers.

5′ End of Probes
Primer Express software does not select probes with a G on the 5′ end. The
quenching effect of a G base in this position will be present even after probe
cleavage. The presence of a G base can result in reduced fluorescence values (ΔRn)
that can negatively affect assay performance. G bases in positions close to the 5′ end,
but not on it, have not been shown to compromise assay performance.

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 Chapter 3 Quantitation Experiments

3′ End of Primers
To reduce the possibility of nonspecific product formation, ensure that the last five
bases on the 3′ end of the primers do not contain more than two C and/or G bases.
Under certain circumstances, such as a G/C-rich template sequence, you may have to
relax this recommendation to keep the amplicon under 150 basepairs in length. In
general, avoid primer 3′ ends extremely rich in G and/or C bases.

Summary of
Probe Guidelines Primer Guidelines
Primer and MGB
Probe Design Select the probe first, then design the primers as close as possible to the probe without
Guidelines overlapping the probe (amplicons of 50 to 150 basepairs are strongly recommended).

Keep the G/C content in the 30 to 80% range.

Avoid runs of an identical nucleotide, especially guanine, where runs of four or more Gs
should be avoided.

When using Primer Express® Software, the When using Primer Express® Software, the
Tm should be 68 to 70 °C. Tm should be 58 to 60 °C.

No G on the 5′ end. The five nucleotides at the 3′ end should

have no more than two G and/or C bases.
Make TaqMan® MGB probes as short as
possible without being shorter than
13 nucleotides.

Applied Biosystems StepOne™ and StepOnePlus™ Real-Time PCR Systems Reagent Guide 3-23
Chapter 3 Quantitation Experiments

Select the Reagents

Several TaqMan and SYBR Green reagents are available for quantitation
experiments. The reagents you use depend on your target:
• DNA or cDNA (below).
• RNA using 1-step RT-PCR (page 3-25).
• RNA using 2-step RT-PCR (page 3-26).
Note: If you are using SYBR Green reagents, Applied Biosystems highly
recommends the Power SYBR Green reagents.

DNA or cDNA
Reagent Kit Part Number
Quantitation
TaqMan® reagents TaqMan® Gene Expression Master Mix, 1-Pack 4369016
(1 × 5 mL), 200 reactions

TaqMan® Gene Expression Master Mix, 10-Pack 4369542

(10 × 5 mL), 2000 reactions

TaqMan® Fast Universal PCR Master Mix (2✕), No 4352042

AmpErase® UNG, 250 Reactions

TaqMan® 2✕ Universal PCR Master Mix, 200 4304437

Reactions

TaqMan® 2✕ Universal PCR Master Mix, 2000 4326708

Reactions

10-Pack, TaqMan® 2✕ Universal PCR Master Mix 4305719

TaqMan® 2✕ Universal PCR Master Mix, No 4324018

AmpErase® UNG, 200 Reactions

TaqMan® 2✕ Universal PCR Master Mix, No 4326614

AmpErase® UNG, 2000 Reactions

10-Pack, TaqMan® 2✕ Universal PCR Master Mix, 4324020

No AmpErase® UNG

TaqMan® PCR Core Reagents Kit, 200 Reactions N808-0228

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 Chapter 3 Quantitation Experiments

Reagent Kit Part Number

SYBR® Green Power SYBR® Green PCR Master Mix (1 mL), 4368577
reagents 40 reactions

Power SYBR® Green PCR Master Mix (5-mL), 4367659

200 reactions

Power SYBR® Green PCR Master Mix 4368708

(10 × 5-mL), 2000 reactions

Power SYBR® Green PCR Master Mix (50 mL), 4367660

2000 reactions

SYBR® Green PCR Master Mix (1-mL), 4344463

40 reactions

SYBR® Green PCR Master Mix (5-mL), 4309155

200 reactions

SYBR® Green PCR Master Mix (50-mL), 4334973

2000 reactions

SYBR® Green PCR Core Reagents, 200 Reactions 4304886

RNA Quantitation
Reagent Kit Part Number
Using 1-Step
RT-PCR TaqMan® reagents TaqMan® One-Step RT-PCR Master Mix 4309169
Reagents Kit

TaqMan® EZ RT-PCR Core Reagents N808-0236

Note: Use the TaqMan® EZ RT-PCR Core
Reagents when a high-temperature RT step is
required.

SYBR® Green Power SYBR® Green RT-PCR Reagents Kit 4368711

reagents
SYBR® Green RT-PCR Reagents 4310179

Applied Biosystems StepOne™ and StepOnePlus™ Real-Time PCR Systems Reagent Guide 3-25
Chapter 3 Quantitation Experiments

RNA Quantitation
Reagent Step Kit Part Number
Using 2-Step
RT-PCR TaqMan® PCR step only TaqMan® Gene Expression Master 4369016
reagents Mix, 1-Pack (1 × 5 mL),
200 reactions

TaqMan® Gene Expression Master 4369542

Mix, 10-Pack (10 × 5 mL),
2000 reactions

TaqMan® 2✕ Universal PCR Master 4304437

Mix

TaqMan® Fast Universal PCR Master 4352042

Mix (2✕), No AmpErase® UNG

RT step only High-Capacity cDNA Reverse 4374966

Transcription Kit

TaqMan® Reverse Transcription N808-234

Reagents

Both RT and TaqMan® Gold RT PCR kit N808-232

PCR steps

SYBR® Green PCR step only Power SYBR® Green PCR Master 4367659
reagents Mix

SYBR® Green PCR Master Mix 4309155

RT step only High-Capacity cDNA Reverse 4374966

Transcription Kit

TaqMan® Reverse Transcription N808-234

Reagents

Both RT and Power SYBR® Green RT-PCR 4368711

PCR steps Reagents Kit

SYBR® Green RT-PCR Reagents 4310179

About AmpliTaq The use of the hot-start enzyme AmpliTaq Gold® DNA Polymerase is an integral
Gold DNA part of Applied Biosystems Assay Design Guidelines for both TaqMan and SYBR
Polymerase Green reagents. AmpliTaq Gold DNA Polymerase ensures a robust reaction and it
can dramatically reduce the amount of nonspecific product formation. A further
benefit is the simplification of assay setup, which can be performed at room
temperature.
Note: The DNA polymerase included in the TaqMan Fast Universal PCR Master
Mix (2✕), No AmpErase UNG, is capable of very fast hot-start PCR. The
performance is similar to that of the AmpliTaq Gold DNA Polymerase.

About MultiScribe™ Reverse Transcriptase is a recombinant Moloney Murine Leukemia

MultiScribe Virus (MuLV) Reverse Transcriptase that reverse transcribes RNA into
Reverse complimentary DNA (cDNA).
Transcriptase

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 Chapter 3 Quantitation Experiments

Use the Recommended Thermal Cycling Conditions

Use the thermal cycling conditions recommended for your sample:
• DNA or cDNA (page 3-28).
• RNA using 1-step RT-PCR (page 3-28).
• RNA using 2-step RT-PCR (page 3-29).
Note: Thermal cycling conditions for Fast reagents differ from thermal cycling
conditions for standard reagents.

About the The StepOnePlus instrument contains six independently thermally regulated
VeriFlex™ VeriFlex™ blocks to help you optimize your thermal cycling conditions.
Technology
If you are running your experiment on a StepOnePlus instrument, you can set a
different temperature for one or more of the VeriFlex blocks, creating up to six
different zones for samples, or you can set the same temperature for each of the
VeriFlex blocks.
For more information on using the VeriFlex sample blocks, access the Help from
within the StepOne software by clicking in the toolbar or pressing F1.

Applied Biosystems StepOne™ and StepOnePlus™ Real-Time PCR Systems Reagent Guide 3-27
Chapter 3 Quantitation Experiments

DNA or cDNA For TaqMan Fast Universal PCR Master Mix (2✕), No AmpErase UNG, follow
Quantitation these conditions:

Times and Temperatures

Initial Steps PCR (40 Cycles)

AmpErase® UNG
Activation Melt Anneal/Extend
Activation ‡

HOLD HOLD CYCLE

2 min @ 50 °C 20 sec @ 95 °C 1 sec @ 95 °C 20 sec @ 60 °C

‡ Required only if AmpErase® UNG is added to the reactions.

For TaqMan Gene Expression Master Mix, TaqMan 2✕ Universal PCR Master Mix,
and Power SYBR Green PCR Master Mix, follow these conditions:

Times and Temperatures

Initial Steps PCR (40 Cycles)

AmpliTaq Gold®
AmpErase® UNG
DNA Polymerase Melt Anneal/Extend
Activation ‡
Activation

HOLD HOLD CYCLE

2 min @ 50 °C 10 min @ 95 °C 15 sec @ 95 °C 1 min @ 60 °C

‡ Required only if AmpErase® UNG is added to the reactions or is included in the master mix.

RNA Quantitation For the TaqMan One-Step RT-PCR Master Mix Reagents Kit and Power SYBR
Using 1-Step Green RT-PCR Reagents Kit, follow these conditions:
RT-PCR
Times and Temperatures

Initial Steps PCR (40 Cycles)

AmpliTaq Gold®
Reverse
DNA Polymerase Melt Anneal/Extend
Transcription
Activation

HOLD HOLD 40 CYCLES

30 min @ 48 °C 10 min @ 95 °C 15 sec @ 95 °C 1 min @ 60 °C

Note: These conditions do not apply to the TaqMan EZ RT-PCR Kit. See the
TaqMan® EZ RT-PCR Kit Protocol for the appropriate conditions.

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 Chapter 3 Quantitation Experiments

RNA Quantitation For the High-Capacity cDNA Reverse Transcription Kit, follow these conditions:
Using 2-Step
RT-PCR Step Times and Temperatures

Step 1 Step 2

1. RT Step HOLD HOLD

10 min @ 25 °C 120 min @ 37 °C

After reverse-transcribing the RNA into cDNA (RT step), samples can be stored or used for
the subsequent PCR step described below.

IMPORTANT! For most applications and when large amounts of cDNA are required,
Applied Biosystems recommends 120 minutes at 37 °C for reverse transcription to
achieve optimal conversion.

For TaqMan Fast Universal PCR Master Mix (2✕), No AmpErase UNG, follow
these conditions:

Step Times and Temperatures

Initial Steps PCR (40 Cycles)

AmpliTaq
AmpErase®
2. PCR Step Gold® DNA Anneal/
UNG Melt
Polymerase Extend
Activation ‡
Activation

HOLD HOLD CYCLE

2 min @ 50 °C 20 sec @ 95 °C 1 sec @ 95 °C 20 sec @ 60 °C

‡ Required only if AmpErase® UNG is added to the reactions.

For TaqMan Gene Expression Master Mix, TaqMan 2✕ Universal PCR Master Mix
(with AmpErase UNG), and Power SYBR Green PCR Master Mix, follow these
conditions:

Step Times and Temperatures

Initial Steps PCR (40 Cycles)

AmpliTaq
AmpErase®
2. PCR Step Gold® DNA Anneal/
UNG Melt
Polymerase Extend
Activation
Activation

HOLD HOLD CYCLE

2 min @ 50 °C 10 min @ 95 °C 15 sec @ 95 °C 1 min @ 60 °C

Applied Biosystems StepOne™ and StepOnePlus™ Real-Time PCR Systems Reagent Guide 3-29
Chapter 3 Quantitation Experiments

Optimize Primer Concentrations

By independently varying forward and reverse primer concentrations, you can
identify the concentrations that provide optimal assay performance. Primers are
always in large molar excess during the exponential phase of PCR amplification.
When you use TaqMan Gene Expression Master Mix or TaqMan 2✕ Universal PCR
Master Mix, Applied Biosystems recommends the primer concentrations listed in
“Default Primer Concentrations” below. Detailed discussions follow for the:
• Primer optimization matrix (below)
• TaqMan reagents (below)
• SYBR Green reagents (page 3-31)

Default Primer The recommended starting primer concentrations listed in the table below are for
Concentrations DNA and cDNA quantitation assays.

Concentrations (nM)
Reagent
Forward Primer Reverse Primer

TaqMan® reagents 900 900

SYBR® Green reagents 50 50

Primer A primer optimization matrix allows you to determine that the minimum primer
Optimization concentration yields the minimum CT and maximum ΔRn.
Matrix
A primer optimization matrix can help to compensate for nonspecific primer binding,
which can reduce the amount of primer available to bind at its specific site.

TaqMan For quantitation assays using TaqMan reagents, you can achieve optimal
Reagents performance by selecting the primer concentrations that provide the lowest CT and
highest ΔRn for a fixed quantity of target template.
Note: Although CT values are the parameter by which quantitative values are
assigned in a real-time quantitation assays, ΔRn values can also be important when
you are trying to obtain maximum sensitivity and reproducibility.
The results of a typical TaqMan reagent primer optimization matrix experiment are
shown in Figure 3-4 on page 3-31:
• Figure 3-4a shows the amplification plots for all primer concentration
combinations in linear view.
• Figure 3-4b shows the same data in log view format.
The combination of 50-nM forward and reverse primer (Plot group C) gives both the
lowest ΔRn and highest CT. All other primer combinations that contain a 150-nM
concentration of either the forward or reverse primer (Plot group B) give a reduced
ΔRn. All primer combinations that contain at least 300-nM forward and reverse
primer (Plot group A) give both the highest ΔRn and the lowest CT; as a result, any of
the plot group A or B provides optimal performance.

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 Chapter 3 Quantitation Experiments

a) Linear view

b) Log view

Figure 3-4 Primer optimization experimental results showing amplification plots

(linear and log views) of primer combinations
Plot group key:
A: Combinations that contain at least 300 nM of forward and reverse primer
B: Combinations that contain at least 150 nM of forward and reverse primer
C: Combinations that contain at least 50 nM of forward and reverse primer

SYBR Green Optimizing primer concentrations is slightly more complex for quantitation assays
Reagents using SYBR Green reagents. You should perform the same primer optimization
matrix as for TaqMan reagents; however, you must include negative controls for
SYBR Green reagents. The primer concentrations you select should provide a low
CT and high ΔRn when run against the target template, but should not produce
nonspecific product formation with negative controls.

Applied Biosystems StepOne™ and StepOnePlus™ Real-Time PCR Systems Reagent Guide 3-31
Chapter 3 Quantitation Experiments

Melt curves or gel analysis can be extremely useful when you select optimal primer
concentrations for quantitation assays using SYBR Green reagents. Figure 3-5 on
page 3-32 shows the results from a primer optimization matrix at primer
concentrations of 900-nM forward and reverse primers:
• Figure 3-5a shows strong amplification of the negative control wells, which
indicates that significant nonspecific amplification is occurring.
• Figure 3-5b shows that the melting temperature of the product generated in the
absence of template is lower than the melting temperature of the specific
product generated with template, indicating that significant nonspecific
amplification is occurring.
The results shown in Figure 3-5 are typical of primer-dimer formation. These results
indicate that lower primer concentrations may provide more optimal results.
Additionally, you can redesign another set of primers to the target of interest.

a) Amplification plot, linear view

Target Amplification

NC (nonspecific
amplification)

b) Melt curve analysis

Target
NC Amplification
(non-specific
amplification)

Figure 3-5 Amplification data using SYBR® Green reagents

(a) Amplification plot (linear view) demonstrating suspected nonspecific
amplification in negative control (NC) wells
(b) Melt curve analysis confirming that product in NC wells has a different melting
temperature from the specific product

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 Chapter 3 Quantitation Experiments

Optimize the Probe Concentration

For detection by TaqMan® probes, the recommended probe concentration of 250 nM
ensures excellent assay performance. However, depending on the requirements of the
assay, a probe optimization experiment can prove useful.
Note: No probe is required for SYBR Green I dye detection.

Recommended The recommended probe concentrations for DNA and cDNA quantitation assays
Probe using TaqMan reagents is 250 nM. Figure 3-6 shows the results of a probe
Concentrations optimization experiment in which the probe concentration is varied from
50 to 250 nM:
• Figure 3-6a shows an increase in ΔRn as the probe concentration is increased.
• Figure 3-6b shows that the CT value changes with sufficient probe
concentrations.
To ensure the best reproducibility, especially when you want to detect low copy
numbers of a target, avoid probe limiting concentrations. Run the assay at a probe
concentration of 250 nM. Using a 250-nM concentration, you avoid probe limitation
and ensure large ΔRn values. Large ΔRn values indicate a robust assay that is
performing at high efficiency, giving high product yield and allowing accurate peak
measurement.

Applied Biosystems StepOne™ and StepOnePlus™ Real-Time PCR Systems Reagent Guide 3-33
Chapter 3 Quantitation Experiments

a) Linear view

b) Log view

Figure 3-6 Amplification plot (linear and log views) of probe concentration
titration from 50 to 250 nM

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 Chapter 3 Quantitation Experiments

For More Information

For information on:
• Using the TaqMan reagents, refer to:
– TaqMan® Fast Universal PCR Master Mix (2✕) Protocol
– TaqMan® Gene Expression Master Mix Protocol
– TaqMan® Universal PCR Master Mix Protocol
• Using the SYBR Green reagents, refer to:
– Power SYBR® Green PCR Master Mix and RT-PCR Protocol
– SYBR® Green PCR Master Mix and RT-PCR Reagents Protocol
– SYBR® Green PCR and RT-PCR Reagents Protocol
• Performing quantitation experiments on the StepOne and StepOnePlus systems,
refer to:
– Applied Biosystems StepOne™ and StepOnePlus™ Real-Time PCR Systems
Getting Started Guide for Standard Curve Experiments
– Applied Biosystems StepOne™ and StepOnePlus™ Real-Time PCR Systems
Getting Started Guide for Relative Standard Curve and Comparative CT
Experiments
• Performing presence/absence experiments on the StepOne and StepOnePlus
systems, refer to the Applied Biosystems StepOne™ and StepOnePlus™
Real-Time PCR Systems Getting Started Guide for Presence/Absence
Experiments.

Applied Biosystems StepOne™ and StepOnePlus™ Real-Time PCR Systems Reagent Guide 3-35
Chapter 3 Quantitation Experiments

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Genotyping Experiments 4 4
This chapter covers:
Section 4.1 About Genotyping Experiments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4-3
Section 4.2 Design Guidelines . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4-9

Applied Biosystems StepOne™ and StepOnePlus™ Real-Time PCR Systems Reagent Guide 4-1
Chapter 4 Genotyping Experiments

4-2 Applied Biosystems StepOne™ and StepOnePlus™ Real-Time PCR Systems Reagent Guide
 Chapter 4 Genotyping Experiments

Section 4.1 About Genotyping Experiments

This section covers:

Overview . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4-4
Select the Assay Type . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4-6

Applied Biosystems StepOne™ and StepOnePlus™ Real-Time PCR Systems Reagent Guide 4-3
Chapter 4 Genotyping Experiments

Overview
What Is a A genotyping experiment is an endpoint experiment used to determine the genotype
Genotyping of unknown samples. With this experiment type, you can differentiate a single
Experiment? nucleotide polymorphism (SNP).
A genotyping experiment determines if unknown samples are:
• Homozygotes (samples having only allele 1)
• Homozygotes (samples having only allele 2)
• Heterozygotes (samples having both allele 1 and allele 2)

Components
PCR reactions for genotyping experiments include the following components:
• Sample – The sample in which the genotype of the target is unknown.
• (Optional) Replicates – Identical reactions containing identical components
and volumes.
• Negative Controls – Samples that contain water or buffer instead of template;
also known as no template controls (NTCs). Negative controls should not
amplify.
• (Optional) Positive controls – Samples that contain known genotypes
(homozygotes for allele 1, homozygotes for allele 2, and heterozygotes for
alleles 1 and 2).

Instruments Genotyping experiments require two steps: thermal cycling (PCR amplification)
followed by endpoint detection of the resulting fluorescent signals. You can perform
the thermal cycling step (PCR amplification) on the Applied Biosystems StepOne™
and StepOnePlus™ Real-Time PCR Systems or on a standalone thermal cycler.
If you use the StepOne™ and StepOnePlus™ systems:
• You can analyze the PCR, which is helpful for troubleshooting.
• Perform the endpoint plate read separately.

How Genotyping In genotyping experiments, the PCR includes a specific fluorescent-dye-labeled

Experiments probe for each allele. The probes contain different fluorescent reporter dyes to
Work differentiate each allele.
You can use TaqMan® minor groove binder (MGB) probes on the StepOne and
StepOnePlus systems. Each TaqMan® MGB probe contains:
• A reporter dye at the 5′ end of each probe
– VIC® dye is linked to the 5′ end of the Allele 1 probe
– FAM™ dye is linked to the 5′ end of the Allele 2 probe
• A minor groove binder (MGB)
This modification increases the melting temperature (Tm) of probes without
increasing probe length (Afonina et al., 1997; Kutyavin et al., 1997), thereby
allowing the design of shorter probes. Consequently, the TaqMan MGB probes
exhibit greater differences in Tm values between matched and mismatched
probes; greater differences in Tm values provide accurate genotyping.

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 Chapter 4 Genotyping Experiments

• A nonfluorescent quencher (NFQ) at the 3′ end of the probe

Because the quencher does not fluoresce, real-time PCR systems can measure
reporter dye contributions more accurately.
During PCR, each probe anneals specifically to its complementary sequence between
the forward and reverse primer sites. AmpliTaq Gold ® DNA polymerase can cleave
only probes that hybridize to the allele sequence (match). Cleavage separates the
reporter dye from the quencher dye, increasing fluorescence of the reporter dye.
Thus, the fluorescence signals generated during PCR amplification indicate the
alleles that are present in the sample.

Mismatches Between Probe and Allele Sequences

Mismatches between a probe and allele (Figure 4-1) reduce the efficiency of probe
hybridization. Furthermore, AmpliTaq Gold DNA polymerase is likely to displace
the mismatched probe rather than to cleave it to release reporter dye.

Allele F
1 V Legend
Q Q
V VIC® dye

Match Mismatch
F FAM™ dye

Q Quencher
Allele F V
2
Q Q AmpliTaq
Gold® DNA
Polymerase

Match Mismatch GR1556

Figure 4-1 Results from matches and mismatches between allele and probe
sequences in genotyping experiments

Table 4-1 summarizes the possible results of the genotyping experiment example
shown above.
Table 4-1 Genotyping experiment results

A substantial increase in… Indicates…

VIC® dye fluorescence only homozygosity for Allele 1.

FAM™ dye fluorescence only homozygosity for Allele 2.

both fluorescent signals heterozygosity.

Workflow Before performing genotyping experiments on the StepOne and StepOnePlus

systems, prepare for the experiment as follows:
1. Select the assay type (below).
2. Review the design guidelines for the assay type you selected (Section 4.2 on
page 4-9).

Applied Biosystems StepOne™ and StepOnePlus™ Real-Time PCR Systems Reagent Guide 4-5
Chapter 4 Genotyping Experiments

Select the Assay Type

When you design your experiments with the StepOne software, you can select the
following assay types for genotyping experiments:
• Pre-Designed/Validated (below)
• Custom (page 4-7)
This section lists the products available for each assay type.
Note: The assays are specific to the target of interest. The master mixes contain the
remaining components needed for the PCR reaction.

Pre-Designed/
Product Attributes
Validated Assays
TaqMan® SNP • Predesigned assays for high-density, genome-wide
Genotyping Assays marker coverage.
• For screening, association, candidate region, candidate
gene, or fine-mapping studies.
• Convenient single-tube format.

TaqMan® Drug • Detect polymorphisms in 220 genes that code for various
Metabolism Genotyping drug metabolism enzymes and drug transporters.
Assays • For studying single-nucleotide polymorphisms (SNPs),
insertions/deletions (in/dels), and multinucleotide
polymorphisms (MNPs).

Pre-Developed TaqMan® • Genotype purified DNA samples for specific mutations;

Assay Reagents for most assays discriminate between two alleles of single-
Allelic Discrimination nucleotide polymorphisms (SNPs).
• Minor groove binder (MGB) added for better genotyping.
• Allele 1 and 2 control DNA included to allow each
homozygote signal to be generated on each run.
• Closed-tube system requires no post-PCR manipulation or
gels.

TaqMan® Genotyping Optimized for endpoint fluorescence detection in SNP

Master Mix genotyping applications:
• Distinct clusters and high call rates for unambiguous allelic
discrimination.
• Excellent pre- and post-PCR stability for high-throughput
setup and analysis.
• Validated with TaqMan® SNP Genotyping Assays.
• Simplifies assay implementation by using one reagent for
all assays.

TaqMan® 2✕ Universal • Provides optimal performance for TaqMan® assays that

PCR Master Mix use cDNA or DNA as a template.
(with or without • Contains components that ensure excellent assay
AmpErase® UNG) performance.
• Simplifies assay implementation by using one reagent for
all assays.

Note: Genotyping experiments are not supported for Fast or SYBR® Green master
mixes and protocols.

4-6 Applied Biosystems StepOne™ and StepOnePlus™ Real-Time PCR Systems Reagent Guide
 Chapter 4 Genotyping Experiments

For guidelines on designing your experiments with Pre-Designed/Validated assays,

see page 4-10.

Custom Assays
Product Attributes

Custom TaqMan® SNP • Any possible single-nucleotide polymorphism (SNP) in any

Genotyping Assays organism.
• Detect insertions/deletions (in/dels) of up to six bases.
• Detect multiple nucleotide polymorphisms (MNPs) of up to
six bases.
• Convenient single-tube format.

TaqMan® Genotyping Optimized for endpoint fluorescence detection in SNP

Master Mix genotyping applications:
• Distinct clusters and high call rates for unambiguous allelic
discrimination.
• Excellent pre- and post-PCR stability for high-throughput
setup and analysis.
• Validated with TaqMan® SNP Genotyping Assays.
• Simplifies assay implementation by using one reagent for
all assays.

TaqMan® 2✕ Universal • Provides optimal performance for TaqMan® assays that

PCR Master Mix use cDNA or DNA as a template.
(with or without • Contains components that ensure excellent assay
AmpErase® UNG) performance.
• Simplifies assay implementation by using one reagent for
all assays.

Note: Genotyping experiments are not supported for Fast or SYBR® Green master
mixes and protocols.
For guidelines on designing your experiments with Custom assays, see page 4-14.

Applied Biosystems StepOne™ and StepOnePlus™ Real-Time PCR Systems Reagent Guide 4-7
Chapter 4 Genotyping Experiments

4-8 Applied Biosystems StepOne™ and StepOnePlus™ Real-Time PCR Systems Reagent Guide
 Chapter 4 Genotyping Experiments

Section 4.2 Design Guidelines

This section covers:

Pre-Designed/Validated Assays. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4-10
TaqMan® SNP Genotyping Assays . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4-10
TaqMan® Drug Metabolism Genotyping Assays . . . . . . . . . . . . . . . . . . . . 4-11
Pre-Developed TaqMan® Assay Reagents for Allelic Discrimination . . . . 4-12
Select the Master Mix . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4-13
Design the Experiment . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4-14
Custom Assays . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4-14
Custom TaqMan® SNP Genotyping Assays . . . . . . . . . . . . . . . . . . . . . . . . 4-14
Select the Master Mix . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4-16
Design the Experiment . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4-16

Applied Biosystems StepOne™ and StepOnePlus™ Real-Time PCR Systems Reagent Guide 4-9
Chapter 4 Genotyping Experiments

Pre-Designed/Validated Assays
Workflow If you are designing a genotyping experiment using Applied Biosystems Pre-
Designed/Validated assays, Applied Biosystems recommends that you follow the
workflow below:
1. Select the assay:
• TaqMan® SNP Genotyping Assays (below).
• TaqMan® Drug Metabolism Genotyping Assays (page 4-11).
• TaqMan® Pre-Developed Assays Reagents for Allelic Discrimination
(page 4-12).
2. Select the master mix (page 4-13).
3. Design the experiment using the StepOne™ software (page 4-14).

TaqMan® SNP Genotyping Assays

Product TaqMan® SNP Genotyping Assays are a comprehensive collection of primer and
Description probe sets for genotyping single nucleotide polymorphisms (SNPs) for human
studies.
The assays:
• Use TaqMan® reagents to amplify and detect specific SNP alleles in purified
genomic DNA samples.
• Are designed using Applied Biosystems bioinformatics pipeline and software,
as well as genomic information from Celera Genomics and public databases.
• Are designed and optimized to work with an Applied Biosystems TaqMan®
master mix, using universal thermal cycling conditions.

Product All TaqMan SNP Genotyping Assays require:

Requirements • Three components:
– 1 to 20 ng of purified genomic DNA sample.
– 20✕, 40✕, or 80✕ SNP Genotyping Assay Mix (specific for each
polymorphism). Each assay consists of sequence-specific forward and
reverse primers to amplify the SNP of interest and two TaqMan MGB
probes: One probe labeled with VIC® dye detects the Allele 1 sequence; one
probe labeled with FAM™ dye detects the Allele 2 sequence.
– TaqMan® Genotyping Master Mix or TaqMan® 2✕ Universal PCR Master
Mix (with or without AmpErase® UNG).
• PCR amplification and an endpoint read to obtain results.

4-10 Applied Biosystems StepOne™ and StepOnePlus™ Real-Time PCR Systems Reagent Guide
 Chapter 4 Genotyping Experiments

Available Assays TaqMan SNP Genotyping Assays are available as TaqMan® Pre-Designed SNP
Genotyping Assays:
• Over 4.5 million predesigned genome-wide assays, including 3.5 million human
HapMap SNPs, 70,000 human cSNPs, 160,000 human validated, and 10,000
mouse assays.
• Available in small, medium, and large scale.
• Made to Order (that is, manufactured at the time of order).

For More • For information on the latest available products and specific product uses, refer
Information to the Applied Biosystems Web sites:
http://www.allsnps.com/
and/or
http://www.appliedbiosystems.com/
a. On the Home page, under TaqMan® Products, select TaqMan® SNP
Genotyping Assays.
b. On the SNP Genotyping Assays page, under Pre-Designed/Validated
Assays, select TaqMan® SNP Genotyping Assays.
• For information on preparing PCR reactions using the TaqMan SNP Genotyping
Assays, refer to the TaqMan® SNP Genotyping Assays Protocol.

TaqMan® Drug Metabolism Genotyping Assays

Product TaqMan® Drug Metabolism Genotyping Assays are a comprehensive collection of

Description Inventoried primer and probe sets for genotyping SNPs, insertions and deletions
(indels), and multiple nucleotide polymorphisms (MNPs) in drug metabolism related
genes.
The assays:
• Use TaqMan reagents to amplify and detect specific polymorphisms in purified
genomic DNA samples.
• Are designed using Applied Biosystems bioinformatics pipeline and software,
as well as genomic information from public SNP databases and public genome
assemblies.
• Are designed and optimized to work with Applied Biosystems TaqMan® master
mixes.

Product All TaqMan Drug Metabolism Genotyping Assays require:

Requirements • Three components:
– 3 to 30 ng of purified genomic DNA sample.
– 20✕ Drug Metabolism Genotyping Assay Mix (specific for each
polymorphism). Each assay consists of sequence-specific forward and
reverse primers to amplify the polymorphic sequence of interest and two
TaqMan MGB probes: One probe labeled with VIC dye detects the Allele 1
sequence; one probe labeled with FAM dye detects the Allele 2 sequence.
– TaqMan® Genotyping Master Mix or TaqMan® 2✕ Universal PCR Master
Mix (with or without AmpErase® UNG).

Applied Biosystems StepOne™ and StepOnePlus™ Real-Time PCR Systems Reagent Guide 4-11
Chapter 4 Genotyping Experiments

• PCR amplification and an endpoint read to obtain results.

For More • For information on the latest available products and specific product uses, refer
Information to the Applied Biosystems Web sites:
http://www.allsnps.com/
and/or
http://www.appliedbiosystems.com/
a. On the Home page, under TaqMan® Products, select TaqMan® SNP
Genotyping Assays.
b. On the SNP Genotyping Assays page, under Pre-Designed/Validated
Assays, select TaqMan® Drug Metabolism Genotyping Assays.
• For information on preparing PCR reactions using the TaqMan SNP Genotyping
Assays, refer to the TaqMan® Drug Metabolism Genotyping Assays Protocol.

Pre-Developed TaqMan® Assay Reagents for Allelic Discrimination

Product Pre-Developed TaqMan® Assay Reagents for Allelic Discrimination (TaqMan®

Description PDARs for AD) are Inventoried assays optimized for the discrimination of specific
alleles.
The assays:
• Use TaqMan reagents to amplify and detect specific polymorphisms in purified
genomic DNA samples.
• Are designed and optimized to work with an Applied Biosystems TaqMan®
master mix, using universal thermal cycling conditions.

Product TaqMan PDARs for AD require three components:

Requirements • 2 to 20 ng of purified genomic DNA sample.
• 10✕ Allelic Discrimination Assay Mix (specific for each polymorphism). Each
assay consists of sequence-specific forward and reverse primers to amplify the
polymorphic sequence of interest and two TaqMan MGB probes: One probe
labeled with VIC dye detects the Allele 1 sequence; one probe labeled with
FAM dye detects the Allele 2 sequence.
• TaqMan® Genotyping Master Mix or TaqMan® 2✕ Universal PCR Master Mix
(with or without AmpErase® UNG).
Note: Allele 1 and 2 control DNA is included with each assay to allow each
homozygote signal to be generated on each run.

For More • For information on the latest available products and specific product uses, refer
Information to the Applied Biosystems Web sites:
http://www.allsnps.com/
and/or
http://www.appliedbiosystems.com/
a. On the Home page, under TaqMan® Products, select TaqMan® SNP
Genotyping Assays.

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 Chapter 4 Genotyping Experiments

b. On the SNP Genotyping Assays page, under Pre-Designed/Validated

Assays, select TaqMan® Pre-Developed Assay Reagents for Allelic
Discrimination (TaqMan® PDARs for AD).
• For information on preparing PCR reactions using the TaqMan PDARs for
Allelic Discrimination, refer to the Pre-Developed TaqMan® Assay Reagents
Allelic Discrimination Protocol.

Select the Master Mix

Available Master Applied Biosystems Pre-Designed/Validated assays for genotyping experiments are
Mixes designed to work with the following master mixes:
• TaqMan PDARs for AD contain TaqMan® 2✕ Universal PCR Master Mix
(with AmpErase® UNG).
• TaqMan SNP Genotyping Assays and Custom TaqMan SNP Genotyping
Assays can be used with:

Master Mix Part Number

TaqMan® Genotyping Master Mix, 1-Pack (1 × 10 mL), 4371355

400 reactions

TaqMan® Genotyping Master Mix, 1 Bulk Pack (1 × 50 mL), 4371357

2000 reactions

TaqMan® 2✕ Universal PCR Master Mix, 200 Reactions 4304437

TaqMan® 2✕ Universal PCR Master Mix, 2000 Reactions 4326708

10-Pack, TaqMan® 2✕ Universal PCR Master Mix 4305719

TaqMan® 2✕ Universal PCR Master Mix, No AmpErase® 4324018

UNG, 200 Reactions

TaqMan® 2✕ Universal PCR Master Mix, No AmpErase® 4326614

UNG, 2000 Reactions

10-Pack, TaqMan® 2✕ Universal PCR Master Mix, No 4324020

AmpErase® UNG

Note: Genotyping experiments are not supported for Fast or SYBR® Green master
mixes and protocols.

For More For information on using the TaqMan master mixes, refer to the:
Information • TaqMan® Genotyping Master Mix Protocol
• TaqMan® Universal PCR Master Mix Protocol

Applied Biosystems StepOne™ and StepOnePlus™ Real-Time PCR Systems Reagent Guide 4-13
Chapter 4 Genotyping Experiments

Design the Experiment

Use the StepOne For Applied Biosystems Pre-Designed/Validated assays, use the StepOne software to
Software design your genotyping experiments. The StepOne software automatically calculates
volumes for the:
• Reaction mix components
• Sample dilutions
Note: To select a SNP assay in the StepOne software, go to the SNP Assays screen in
the Design Wizard or to the Plate Setup screen in Advanced Setup. In the SNP
Assays screen or Plate Setup screen, you can select an assay from the library or
create a new assay.

For More For information on designing and performing genotyping experiments on the
Information StepOne and StepOnePlus systems, refer to Applied Biosystems StepOne™ and
StepOnePlus™ Real-Time PCR Systems Getting Started Guide for Genotyping
Experiments.

Custom Assays
Workflow If you are designing a genotyping experiment using Applied Biosystems Custom
assays, Applied Biosystems recommends that you follow the workflow below:
1. Order the assay: Custom TaqMan® SNP Genotyping Assays (below).
2. Select the master mix (page 4-16).
3. Design the experiment using the StepOne software (page 4-16).

Custom TaqMan® SNP Genotyping Assays

Product Custom TaqMan® SNP Genotyping Assays are TaqMan probe and primer sets that
Description are designed, synthesized, and formulated by the Custom TaqMan® Genomic Assays
service based on sequence information that you submit. Custom TaqMan SNP
Genotyping Assays allow you to:

Action Example

Perform genotyping studies with any AGTTCATCCATGGTCA -->

possible single-nucleotide polymorphism AGTTCATACATGGTCA
(SNP) in any organism
Annotated as: AGTTCAT[C/A]CATGGTCA

Detect insertions/deletions (in/dels) of up to AGTTCATCCATGGTCA -->

six bases for genotyping studies AGTTCATGGTCA
Annotated as: AGTTCAT[CCAT/*]GGTCA

Detect multiple nucleotide polymorphisms AGTTCATCCGGTCA -->

(MNPs) of up to six bases for genotyping AGTTCATATGGTCA
studies
Annotated as: AGTTCAT[CC/AT]GGTCA

4-14 Applied Biosystems StepOne™ and StepOnePlus™ Real-Time PCR Systems Reagent Guide
 Chapter 4 Genotyping Experiments

The assays:
• Use TaqMan reagents to amplify and detect specific polymorphisms in purified
genomic DNA (gDNA).
• Are developed using proprietary assay-design software.
• Are designed and optimized to work with an Applied Biosystems TaqMan®
master mix, using universal thermal cycling conditions.

Product All Custom TaqMan SNP Genotyping Assays require:

Requirements • A submission file that includes your target SNP sequence. You create the
submission file using free File Builder software, then submit the file to the
Custom TaqMan® Genomic Assays service.
• Three components:
– 1 to 20 ng of purified gDNA sample per well.
– 40✕ SNP Genotyping Assay or 80✕ SNP Genotyping Assay (specific for
each polymorphism). Each assay consists of sequence-specific forward and
reverse primers to amplify the SNP of interest and two TaqMan MGB
probes: One probe labeled with VIC dye detects the Allele 1 sequence; one
probe labeled with FAM dye detects the Allele 2 sequence.
– TaqMan® Genotyping Master Mix or TaqMan® Universal PCR Master Mix
(with or without AmpErase® UNG).
• PCR amplification and an endpoint read to obtain results.

For More • For information on the latest available products and specific product uses, refer
Information to the Applied Biosystems Web sites:
http://www.allsnps.com/
and/or
http://www.appliedbiosystems.com/
a. On the Home page, under TaqMan® Products, select TaqMan® SNP
Genotyping Assays.
b. On the SNP Genotyping Assays page, under Custom Assays, select
Custom TaqMan® SNP Genotyping Assays.
• For information on ordering Custom TaqMan SNP Genotyping Assays, refer to
the Custom TaqMan® Genomic Assays Protocol: Submission Guidelines.
• For information on preparing PCR reactions using the Custom TaqMan SNP
Genotyping Assays, refer to the Custom TaqMan® SNP Genotyping Assays
Protocol.

Applied Biosystems StepOne™ and StepOnePlus™ Real-Time PCR Systems Reagent Guide 4-15
Chapter 4 Genotyping Experiments

Select the Master Mix

Available Master Applied Biosystems Made to Order assays for genotyping experiments are designed
Mixes to work with the following master mixes:

Master Mix Part Number

TaqMan® Genotyping Master Mix, 1-Pack (1 × 10 mL), 400 reactions 4371355

TaqMan® Genotyping Master Mix, 1 Bulk Pack (1 × 50 mL), 4371357

2000 reactions

TaqMan® 2✕ Universal PCR Master Mix, 200 Reactions 4304437

TaqMan® 2✕ Universal PCR Master Mix, 2000 Reactions 4326708

10-Pack, TaqMan® 2✕ Universal PCR Master Mix 4305719

TaqMan® 2✕ Universal PCR Master Mix, No AmpErase® UNG, 200 4324018

Reactions

TaqMan® 2✕ Universal PCR Master Mix, No AmpErase® UNG, 2000 4326614

Reactions

10-Pack, TaqMan® 2✕ Universal PCR Master Mix, No AmpErase® 4324020

UNG

Note: Genotyping experiments are not supported for Fast or SYBR® Green master
mixes and protocols.

For More For information on using the TaqMan master mixes, refer to the:
Information • TaqMan® Genotyping Master Mix Protocol
• TaqMan® Universal PCR Master Mix Protocol

Design the Experiment

Use the StepOne For Applied Biosystems Custom assays, use the StepOne software to design your
Software genotyping experiments. The StepOne software automatically calculates volumes for
the:
• Reaction mix components
• Sample dilutions
Note: To select a SNP assay in the StepOne software, go to the SNP Assays screen
in the Design Wizard or to the Plate Setup screen in Advanced Setup. In the SNP
Assays screen or Plate Setup screen, you can select an assay from the library or
create a new assay.

For More For information on designing and performing genotyping experiments on the
Information StepOne and StepOnePlus systems, refer to Applied Biosystems StepOne™ and
StepOnePlus™ Real-Time PCR Systems Getting Started Guide for Genotyping
Experiments.

4-16 Applied Biosystems StepOne™ and StepOnePlus™ Real-Time PCR Systems Reagent Guide
Presence/Absence Experiments 5 5
This chapter covers:
Section 5.1 About Presence/Absence Experiments. . . . . . . . . . . . . . . . . . . . . . . . . 5-3
Section 5.2 Design Guidelines . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5-9

Applied Biosystems StepOne™ and StepOnePlus™ Real-Time PCR Systems Reagent Guide 5-1
Chapter 5 Presence/Absence Experiments

5-2 Applied Biosystems StepOne™ and StepOnePlus™ Real-Time PCR Systems Reagent Guide
 Chapter 5 Presence/Absence Experiments

Section 5.1 About Presence/Absence Experiments

This section covers:

Overview . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5-4
Select the Assay Type . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5-6

Applied Biosystems StepOne™ and StepOnePlus™ Real-Time PCR Systems Reagent Guide 5-3
Chapter 5 Presence/Absence Experiments

Overview
What Is a A presence/absence experiment is an endpoint experiment that indicates the presence
Presence/ or absence of a specific nucleic acid sequence (target) in a sample. The actual
Absence quantity of target is not determined.
Experiment?
Presence/absence experiments are commonly used to detect the presence or absence
of a pathogen, such as a viral or bacterial pathogen. For example, a presence/absence
experiment might be used to determine if Salmonella bacteria are present in
hamburger meat. The results will show if Salmonella bacteria are present or are not
present; the quantity of bacteria is not determined.

Components
PCR reactions for presence/absence experiments include the following components:
• Sample – The sample in which the presence of a target is unknown.
• Replicates – Identical reactions containing identical components and volumes.
• Internal positive control (IPC) – A short synthetic DNA template that is added
to PCR reactions. You can use the IPC to distinguish between true negative
results and reactions affected by PCR inhibitors, incorrect assay setup, or a
reagent or instrument failure.
Note: Presence/absence experiments can be performed without an IPC;
however, the IPC ensures that a failed PCR is not mistaken for a negative test
result.
• Negative Controls – Wells that contain water or buffer instead of sample
template. No amplification of the target should occur in negative control wells.
In the StepOne software, you can set up the PCR reactions for the presence/absence
experiments three different ways:

Setup Well Types

IPC setup There are three types of wells:

• Unknown-IPC wells – Wells contain sample template and IPC
template; the presence of the target is not known.
• Negative control-IPC wells – Wells contain IPC template and water
or buffer instead of sample template in the PCR reaction. Only the
IPC template should amplify in negative control-IPC wells because
the reaction contains no sample template. Also called IPC+.
• Negative control-blocked IPC wells – Wells contain IPC blocking
agent instead of sample template in the PCR reaction. No
amplification should occur in negative control-blocked IPC wells
because the reaction contains no sample template and amplification
of the IPC is blocked. Also called no amplification control (NAC).

No IPC, There are two types of wells:

singleplex
• Unknown wells – Wells contain sample template; the presence of
setup
the target is not known.
• Negative controls –Wells contain water or buffer instead of sample
template.

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 Chapter 5 Presence/Absence Experiments

Setup Well Types

No IPC, There are two types of wells:

multiplex
• Unknown-Unknown wells – Wells contain sample template; the
setup
presence of the target is not known.
• Negative control-Negative control wells – Wells contain water or
buffer instead of sample template.

Endpoint Presence/absence experiments are endpoint experiments in which fluorescence data

Detection and are collected after the PCR is complete.
Post-PCR Plate
To aid in troubleshooting presence/absence experiments, you can use the Applied
Read
Biosystems StepOne™ and StepOnePlus™ Real-Time PCR Systems to perform real-
time PCR. If you use the StepOne™ and StepOnePlus™ systems for PCR
amplification, perform the pre-PCR read and post-PCR read runs separately.

How Presence/ During the PCR, the fluorogenic probes anneal specifically to the complementary
Absence target between the forward and reverse primer sites on the template DNA. Then
Experiments during extension, AmpliTaq Gold® DNA polymerase cleaves the hybridized probes
Work in each sample containing the target. The cleavage of each matched probe separates
the reporter dye from the quencher dye, resulting in increased fluorescence by the
reporter.
After PCR cycling, the StepOne™ and StepOnePlus™ instruments read the
fluorescence generated during the PCR amplification. The fluorescent signals are
used to determine the presence or absence of the target in each sample. Reporter
signals are normalized to the emission of a passive reference, as follows:
Rn (TT) = Emission Intensity of Target Template Sequence
Emission Intensity of Passive Reference

Rn (IPC) = Emission Intensity of Internal Positive Control

Emission Intensity of Passive Reference

Incorporating an IPC
An IPC is a second TaqMan® probe and primer set added to the reaction plate to
detect a low-copy, constitutive nucleic acid. The IPC and the target are amplified
simultaneously in the same reaction well. If a well does not exhibit amplification, the
StepOne™ software uses the positive signal from the IPC to confirm that the well
failed to amplify because of a lack of target template, rather than because of a
pipetting error or inhibition.
Note: Presence/absence experiments can be performed without an IPC; however, the
IPC ensures that a failed PCR is not mistaken for a negative test result.

Applied Biosystems StepOne™ and StepOnePlus™ Real-Time PCR Systems Reagent Guide 5-5
Chapter 5 Presence/Absence Experiments

Workflow Before performing presence/absence experiments on the StepOne and StepOnePlus

systems, prepare for the experiment as follows:
1. Select the assay type (below).
2. Review the design guidelines for the assay type you selected (Section 5.2 on
page 5-9).

Select the Assay Type

When you design your experiments with the StepOne software, you can select the
following assay types for presence/absence experiments:
• Inventoried/Made to Order (page 5-7)
• Custom (page 5-8)
This section lists the products available for each assay type.
Note: The assays are specific to the target of interest. The master mixes contain the
remaining components needed for the PCR reaction.

5-6 Applied Biosystems StepOne™ and StepOnePlus™ Real-Time PCR Systems Reagent Guide
 Chapter 5 Presence/Absence Experiments

Inventoried/Made
Product Attributes
to Order Assays
TaqMan® Gene • Predesigned, gene-specific primer and probe sets for
Expression Assays human, mouse, rat, Arabidopsis, Drosophila, C. elegans,
C. familiares (dog), and M. mulatta (Rhesus) genes.
• Probe is a FAM™ dye-labeled MGB probe.
• Provided in a convenient, single, 205 tube.
• Available as Inventoried or Made to Order assays.

TaqMan® Endogenous • Optimized, preformulated, ready-to-use endogenous

Control Assays control assays.
Note: FAM™ dye- • Cost-effective gene expression quantitation for human,
labeled TaqMan® mouse, rat, Arabidopsis, Drosophila, and any eukaryotic
Endogenous Control species.
Assays are included as • Choice of FAM™ dye or VIC® dye labels (primer-limited).
Inventoried TaqMan
Gene Expression Assays.

Custom TaqMan® Gene • Any species or organism.

Expression Assays • Target of your choice.
• Probe is a FAM™ dye-labeled MGB probe.
• Provided in a convenient, single, 205 tube.

TaqMan® Gene Tailored for precise quantitation by real-time PCR for routine
Expression Master Mix and challenging experiments:
• Sensitive detection down to 1 copy of target.
• Multiplex PCR for co-amplifying two targets in a single
reaction.
• Specificity for differentiation between gene family
members.
• Validated with TaqMan® Gene Expression Assays.
• Simplifies assay implementation by using one reagent for
all assays.

TaqMan® 2✕ Universal • Provides optimal performance for TaqMan® assays that

PCR Master Mix use cDNA or DNA as a template.
(with or without • Contains components that ensure excellent assay
AmpErase® UNG) performance.
• Simplifies assay implementation by using one reagent for
all assays.

IMPORTANT! Applied Biosystems does not recommend the use of TAMRA™ dye as
a reporter or quencher with the StepOne system. TAMRA dye may be used as a
reporter or quencher with the StepOnePlus system.
Note: Presence/absence experiments are not supported for Fast or SYBR® Green
master mixes and protocols.
For guidelines on designing your experiments with Inventoried/Made to Order
assays, see page 5-10.

Applied Biosystems StepOne™ and StepOnePlus™ Real-Time PCR Systems Reagent Guide 5-7
Chapter 5 Presence/Absence Experiments

Custom Assays
Product Attributes

Custom TaqMan® Probes • Any species or organism.

and Primers • Choice of dye labels, quenchers, and synthesis scales.
• For use with the Primer Express® Software and Applied
Biosystems Assay Design Guidelines.

Primer Express® Software that designs primers and probes for real-time PCR.
Software

TaqMan® Gene Tailored for precise quantitation by real-time PCR for routine
Expression Master Mix and challenging experiments:
• Sensitive detection down to 1 copy of target.
• Multiplex PCR for co-amplifying two targets in a single
reaction.
• Specificity for differentiation between gene family
members.
• Validated with TaqMan® Gene Expression Assays.
• Simplifies assay implementation by using one reagent for
all assays.

TaqMan® 2✕ Universal • Provides optimal performance for TaqMan® assays that

PCR Master Mix use cDNA or DNA as a template.
(with or without • Contains components that ensure excellent assay
AmpErase® UNG) performance.
• Simplifies assay implementation by using one reagent for
all assays.

IMPORTANT! Applied Biosystems does not recommend the use of TAMRA™ dye as
a reporter or quencher with the StepOne system. TAMRA dye may be used as a
reporter or quencher with the StepOnePlus system.
Note: Presence/absence experiments are not supported for Fast or SYBR® Green
master mixes and protocols.
For guidelines on designing your experiments with Custom assays, see page 5-16.

5-8 Applied Biosystems StepOne™ and StepOnePlus™ Real-Time PCR Systems Reagent Guide
 Chapter 5 Presence/Absence Experiments

Section 5.2 Design Guidelines

This section covers:

Inventoried/Made to Order Assays . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5-10
TaqMan® Gene Expression Assays . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5-10
Custom TaqMan® Gene Expression Assays . . . . . . . . . . . . . . . . . . . . . . . . 5-12
TaqMan® Exogenous Internal Positive Control Reagents . . . . . . . . . . . . . . 5-13
Select the Master Mix . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5-15
Design the Experiment . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5-16
Custom Assays . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5-16

Applied Biosystems StepOne™ and StepOnePlus™ Real-Time PCR Systems Reagent Guide 5-9
Chapter 5 Presence/Absence Experiments

Inventoried/Made to Order Assays

Workflow If you select the Inventoried/Made to Order assay type in the StepOne™ software,
Applied Biosystems recommends that you follow the workflow below:
1. Select the assay:
• TaqMan® Gene Expression Assays (below).
• Custom TaqMan® Gene Expression Assays (page 5-12).
2. Use the TaqMan® Exogenous Internal Positive Control Reagents (page 5-13).
Note: Presence/absence experiments can be performed without an IPC;
however, the IPC ensures that a failed PCR is not mistaken for a negative test
result.
3. Select the master mix (page 5-15).
4. Design the experiment using the StepOne software (page 5-16).

TaqMan® Gene Expression Assays

Product TaqMan® Gene Expression Assays are a comprehensive collection of Inventoried

Description and Made to Order probe and primer sets that you can use to perform
presence/absence experiments on human, mouse, rat, Arabidopsis, Drosophila, C.
elegans, C. familiares (dog), and M. mulatta (Rhesus) genes.
The assays:
• Use TaqMan® reagents to amplify and detect the target in cDNA samples.
• Are designed using an automated design and quality-controlled pipeline.
Inventoried assays are manufactured and placed in inventory; Made to Order
assays are predesigned and manufactured when ordered.
• Are designed and optimized to work with an Applied Biosystems TaqMan®
master mix, using universal thermal cycling conditions.
• When possible, amplify target cDNA without amplifying genomic DNA
(m suffix in assay ID) by designing probes that cross exon-exon junctions.

Product All TaqMan Gene Expression Assays require:

Requirements • Three components:
– 1 to 100 ng of cDNA sample (converted from RNA) per well, with all wells
in a study having the same amount of cDNA.
– 20✕ Gene Expression Assay Mix (specific for each target). Each assay mix
consists of two unlabeled PCR primers and a FAM™ dye-labeled TaqMan®
MGB (minor groove binder) probe in a preformulated 20✕ mix. 1✕ final
concentrations are 250 nM for the probe and 900 nM for each primer.
– TaqMan® Gene Expression Master Mix or TaqMan Universal PCR Master
Mix (with or without AmpErase UNG).

5-10 Applied Biosystems StepOne™ and StepOnePlus™ Real-Time PCR Systems Reagent Guide
 Chapter 5 Presence/Absence Experiments

Available Assays TaqMan Gene Expression Assays are available for human, mouse, rat, Arabidopsis,
Drosophila, C. elegans, C. familiares (dog), and M. mulatta (Rhesus) genes. The part
numbers are:
• PN 4331182 for Inventoried assays
• PN 4351372 for Made to Order assays
The prefix of the assay name indicates the species for which the assay was designed:
Hs for Homo sapiens (human), Mm for Mus musculus (mouse), Rn for Rattus
norvegicus (rat), At for Arabidopsis thaliana, Dm for Drosophila melanogaster,
Ce for C. elegans, Cf for C. familiares (dog), and Rh for M. mulatta (Rhesus).
The suffix of the assay name indicates the assay placement, as described in the table
below.

Suffix Description

_m The assay’s probe spans an exon junction; the assay does not detect
genomic DNA.

_s The assay’s primers and probes are designed within a single exon; the assay
does detect genomic DNA.

_g The assay may detect genomic DNA; the assay’s primers and probes may be
within a single exon.

_mH The assay was designed to a transcript belonging to a gene family with high
sequence homology. The assay provides between 10 CT and 15 CT difference
_sH between the target gene and the gene with the closest sequence homology.
Therefore, the assay detects the target transcript with 1000- to 30,000-fold
_gH greater discrimination (sensitivity) than the closest homologous transcript, if
they are present at the same copy number in a sample.

_u The assay’s amplicon spans an exon junction and the probe sits completely in
one of the spanned exons.

TaqMan® Endogenous Control Assays

TaqMan® Endogenous Control Assays are available as:
• Inventoried TaqMan Gene Expression Assays (PN 4331182) – Each assay
contains a FAM™ dye-labeled TaqMan® MGB probe in a single, preformulated
20✕ tube.
• Individual control assays for all human, mouse, and rat species (various part
numbers) – Each assay contains either a FAM™ dye-labeled TaqMan® MGB
probe, a VIC® dye-labeled TaqMan® MGB probe, or a TAMRA™ dye-labeled
probe. TaqMan Endogenous Controls with VIC dye labels are primer-limited.
IMPORTANT! Applied Biosystems does not recommend the use of TAMRA™ dye as
a reporter or quencher with the StepOne system. TAMRA dye may be used as a
reporter or quencher with the StepOnePlus system.

Applied Biosystems StepOne™ and StepOnePlus™ Real-Time PCR Systems Reagent Guide 5-11
Chapter 5 Presence/Absence Experiments

For More • For information on the latest available products and specific product uses, refer
Information to the Applied Biosystems Web site:
http://www.appliedbiosystems.com/
a. On the Home page, under TaqMan® Products, select TaqMan® Gene
Expression Assays.
b. On the Gene Expression Assays & Arrays page:
• Under Individual Assays, select TaqMan® Gene Expression Assays.
This option links to all TaqMan Gene Expression Assays, including
TaqMan Endogenous Control Assays that contain FAM dye-labeled
probes.
or
• Under Individual Control Assays, select TaqMan® Endogenous
Control Assays. This option links to the individual TaqMan
Endogenous Control Assays (that contain FAM dye-labeled TaqMan
MGB probes, VIC dye-labeled TaqMan MGB probes, or TAMRA
dye-labeled probes).
• For information on Custom TaqMan Endogenous Control Assays, refer to the
Using TaqMan® Endogenous Control Assays to Select an Endogenous Control
for Experimental Studies Application Note.
• For information on preparing PCR reactions using the TaqMan Gene Expression
Assays, refer to the TaqMan® Gene Expression Assays Protocol.

Custom TaqMan® Gene Expression Assays

Product Custom TaqMan® Gene Expression Assays are TaqMan probe and primer sets that
Description are designed, synthesized, and formulated by the Custom TaqMan® Genomic Assays
service based on sequence information that you submit. Custom TaqMan Gene
Expression Assays allow you to perform presence/absence experiments on any gene
or splice variant in any organism.
The assays:
• Use TaqMan® reagents to amplify and detect the target in cDNA samples.
• Are developed using proprietary assay-design software.
• Are designed and optimized to work with an Applied Biosystems TaqMan®
master mix, using universal thermal cycling conditions.

Product All Custom TaqMan Gene Expression Assays require:

Requirements • A submission file that includes your target sequence. You create the submission
file using free File Builder software, then submit the file to the Custom
TaqMan® Genomic Assays service.
• Three components:
– 1 to 100 ng of cDNA sample (converted from RNA) per well, with all wells
in a study having the same amount of cDNA.
– 20✕ Gene Expression Assay or 60✕ Gene Expression Assay (specific for
each target). Each assay consists of two target-specific primers and a FAM™
dye-labeled TaqMan MGB probe in a preformulated 20✕ or 60✕ mix. 1✕
final concentrations are 250 nM for the probe and 900 nM for each primer.

5-12 Applied Biosystems StepOne™ and StepOnePlus™ Real-Time PCR Systems Reagent Guide
 Chapter 5 Presence/Absence Experiments

– TaqMan® Gene Expression Master Mix or TaqMan Universal PCR Master

Mix (with or without AmpErase UNG).

For More • For information on the latest available products and specific product uses, refer
Information to the Applied Biosystems Web site:
http://www.appliedbiosystems.com/
a. On the Home page, under TaqMan® Products, select TaqMan® Gene
Expression Assays.
b. On the Gene Expression Assays & Arrays page, under Individual Assays,
select Custom TaqMan® Gene Expression Assays.
• For information on ordering Custom TaqMan Gene Expression Assays, refer to
the Custom TaqMan® Genomic Assays Protocol: Submission Guidelines.
• For information on preparing PCR reactions using the Custom TaqMan Gene
Expression Assays, refer to the Custom TaqMan® Gene Expression Assays
Protocol.

TaqMan® Exogenous Internal Positive Control Reagents

Product The Applied Biosystems TaqMan® Exogenous Internal Positive Control Reagents
Description contain:
• An internal positive control (IPC) with predesigned primers and probe
• IPC DNA template
• IPC blocking control
The reagents are designed to:
• Distinguish types of negative results:
– A negative call for the target and positive call for the IPC indicates that no
target is present.
– A negative call for the target and negative call for the IPC suggests PCR
inhibition.
• Avoid amplification of endogenous controls.
• Permit co-amplification of the IPC and the target without compromising
amplification of the target.
• Detect the IPC using a VIC® dye-labeled probe.
• Detect the target using a FAM™ dye-labeled probe.
• Work with the TaqMan® 2✕ Universal PCR Master Mix (with or without
AmpErase® UNG), using universal thermal cycling conditions.

Product The TaqMan Exogenous Internal Positive Control Reagents kits require the
Requirements following components:
• DNA sample
• TaqMan assay for your target of interest
• TaqMan 2✕ Universal PCR Master Mix (with or without AmpErase UNG)

Applied Biosystems StepOne™ and StepOnePlus™ Real-Time PCR Systems Reagent Guide 5-13
Chapter 5 Presence/Absence Experiments

Available Kits The following TaqMan Exogenous Internal Positive Control Reagents kits are
available from Applied Biosystems:

Kits Part Number

TaqMan® Exogenous Internal Positive Control Reagents with 4308320

TaqMan® 2✕ Universal PCR Master Mix (with VIC® dye)

TaqMan® Exogenous Internal Positive Control Reagents 4308323

Note: If you are using this kit, you will need to purchase one of these
TaqMan® reagents separately:
• TaqMan® 2✕ Universal PCR Master Mix (PN 4304437)
• TaqMan® PCR Core Reagents Kit (PN N808-0228)

IMPORTANT! The kits listed above contain TAMRA™ dye-labeled probes; Applied
Biosystems does not recommend the use of TAMRA dye as a reporter or quencher
with the StepOne system. The kits may be used with the StepOnePlus system;
TAMRA dye may be used as a reporter or quencher with the StepOnePlus system.

5-14 Applied Biosystems StepOne™ and StepOnePlus™ Real-Time PCR Systems Reagent Guide
 Chapter 5 Presence/Absence Experiments

For More For information on preparing PCR reactions using the TaqMan Exogenous Internal
Information Positive Control Reagents, refer to the TaqMan® Exogenous Internal Positive Control
Reagents Protocol.

Select the Master Mix

Available Master Applied Biosystems Inventoried/Made to Order assays for presence/absence

Mixes experiments are designed to work with the following master mixes:

Master Mix Part Number

TaqMan® Gene Expression Master Mix, 1-Pack (1 × 5 mL), 4369016

200 reactions

TaqMan® Gene Expression Master Mix, 10-Pack (10 × 5 mL), 4369542

2000 reactions

TaqMan® 2✕ Universal PCR Master Mix, 200 Reactions 4304437

TaqMan® 2✕ Universal PCR Master Mix, 2000 Reactions 4326708

10-Pack, TaqMan® 2✕ Universal PCR Master Mix 4305719

TaqMan® 2✕ Universal PCR Master Mix, No AmpErase® UNG, 200 4324018

Reactions

TaqMan® 2✕ Universal PCR Master Mix, No AmpErase® UNG, 2000 4326614

Reactions

10-Pack, TaqMan® 2✕ Universal PCR Master Mix, No AmpErase® 4324020

UNG

TaqMan® PCR Core Reagents Kit N808-0228

Note: If you purchase the TaqMan® Exogenous Internal Positive Control Reagents
with TaqMan® 2✕ Universal PCR Master Mix kit (PN 4308320), you do not need to
purchase the master mix separately.
Note: Presence/absence experiments are not supported for Fast or SYBR® Green
master mixes and protocols.

For More For information on using the TaqMan reagents, refer to the:
Information • TaqMan® Gene Expression Master Mix Protocol
• TaqMan® Universal PCR Master Mix Protocol

Applied Biosystems StepOne™ and StepOnePlus™ Real-Time PCR Systems Reagent Guide 5-15
Chapter 5 Presence/Absence Experiments

Design the Experiment

Use the StepOne For Applied Biosystems Inventoried/Made to Order assays, use the StepOne software
Software to design your presence/absence experiments. The StepOne software automatically
calculates volumes for the:
• Reaction mix components
• Controls and samples
• Sample dilutions
Note: To select the Inventoried/Made to Order assay type in the StepOne software,
go to the Reaction Setup screen in either the Design Wizard or Advanced Setup, then
select Inventoried/Made to Order from the Assay Type dropdown menu.

For More For information on designing and performing presence/absence experiments on the
Information StepOne and StepOnePlus systems, refer to the Applied Biosystems StepOne™ and
StepOnePlus™ Real-Time PCR Systems Getting Started Guide for Presence/Absence
Experiments.

Custom Assays
Workflow If you select the Custom assay type in the StepOne™ software for a presence/absence
experiment (that is, you are designing your own primers and probes), Applied
Biosystems recommends that you follow the workflow for the Applied Biosystems
Assay Design Guidelines:
1. Design primers and probes using Primer Express® Software (page 3-21).
2. Select the appropriate reagents (page 3-24).
IMPORTANT! Applied Biosystems does not recommend the use of TAMRA™
dye as a reporter or quencher with the StepOne system. TAMRA dye may be
used as a reporter or quencher with the StepOnePlus system.
Presence/absence experiments are not supported for Fast or SYBR® Green
master mixes and protocols.
3. Use the recommended thermal cycling conditions (page 3-27).
4. Begin with default primer and probe concentrations. If needed, optimize the
primer concentrations (page 3-30) and probe concentrations (page 3-33).
IMPORTANT! These steps provide a rapid and reliable system for assay design and
optimization only when used in their entirety. Adopt the system as a whole to
achieve the highest level of success. For a more detailed description of Applied
Biosystems Assay Design Guidelines, see Appendix C.
Note: To select the Custom assay type in the StepOne software, go to the Reaction
Setup screen in either the Design Wizard or Advanced Setup, then select Custom
from the Assay Type dropdown menu.

5-16 Applied Biosystems StepOne™ and StepOnePlus™ Real-Time PCR Systems Reagent Guide
Formulas A A
This appendix covers:
Standard Deviation Calculation Using the Standard Curve Method. . . . . . . . . . . A-2
Standard Deviation Calculation Using the Comparative Method . . . . . . . . . . . . . A-5
Formula for Comparative CT (ΔΔCT) Experiments. . . . . . . . . . . . . . . . . . . . . . . . A-7

Applied Biosystems StepOne™ and StepOnePlus™ Real-Time PCR Systems Reagent Guide A-1
Appendix A Formulas

Standard Deviation Calculation Using the Standard Curve

Method
Example Comparing Samples with a Reference Sample
The normalized amount of target (c-mycN in the table below) is a unitless number
that can be used to compare the relative amount of target in different samples. One
way to make this comparison is to designate one of the samples as a reference
sample. In the table below, brain is designated as the reference sample; brain is
arbitrarily chosen because it has the lowest expression level of the target.

Relative Standard Curve Results

Each c-mycN value in the table below is divided by the brain c-mycN value to give
the values in the final column. These results indicate the kidney sample contains 5.5×
as much c-myc mRNA as the brain sample, liver 34.2× as much, and lung 15.7× as
much.
To determine relative values, perform the following steps:
1. Average the c-myc and GAPDH values from the table below.
2. Divide the c-myc average by the GAPDH average.
3. Designate the reference sample.
4. Divide the averaged sample value by the averaged reference sample value.

c-myc GAPDH c-mycN c-mycN

Tissue
ng Total Raji RNA ng Total Raji RNA Norm. to GAPDH ‡ Rel. to Brain §

Brain 0.033 0.51

0.043 0.56

0.036 0.59

0.043 0.53

0.039 0.51

0.040 0.52

Average 0.039±0.004 0.54±0.034 0.07±0.008 1.0±0.12

Kidney 0.40 0.96

0.41 1.06

0.41 1.05

0.39 1.07

0.42 1.06

0.43 0.96

Average 0.41±0.016 1.02±0.052 0.40±0.025 5.5±0.35

A-2 Applied Biosystems StepOne™ and StepOnePlus™ Real-Time PCR Systems Reagent Guide
 Appendix A Formulas

c-myc GAPDH c-mycN c-mycN

Tissue
ng Total Raji RNA ng Total Raji RNA Norm. to GAPDH ‡ Rel. to Brain §

Liver 0.67 0.29

0.66 0.28

0.70 0.28

0.76 0.29

0.70 0.26

0.68 0.27

Average 0.70±0.036 0.28±0.013 2.49±0.173 34.2±2.37

Lung 0.97 0.82

0.92 0.88

0.86 0.78

0.89 0.77

0.94 0.79

0.97 0.80

Average 0.93±0.044 0.81±0.041 1.15±0.079 15.7±1.09

‡ The c-mycN value is determined by dividing the average c-myc value by the average GAPDH value. The standard
deviation of the quotient is calculated from the standard deviations of the c-myc and GAPDH values. See “Formula” on
page A-4.
§ The calculation of c-mycN relative to brain involves division by the reference sample value. This is division by an arbitrary
constant, so the cv of this result is the same as the cv for c-mycN.

Applied Biosystems StepOne™ and StepOnePlus™ Real-Time PCR Systems Reagent Guide A-3
Appendix A Formulas

Formula The c-mycN value is determined by dividing the average c-myc value by the average
GAPDH value. The standard deviation of the quotient is calculated from the standard
deviations of the c-myc and GAPDH values using the following formula:

2
cv = cv1 + cv 22

where:

cv = --s- = -------------------------
stddev -
X meanvalue

Using the brain sample from table on page A-2 as an example:

0.004
cv 1 = -------------
0.039

and

0.034
cv 2 = -------------
0.54

2 2
cv = ⎛ 0.004 0.034
-------------⎞ + ⎛ -------------⎞ = 0.12
⎝ 0.039⎠ ⎝ 0.54 ⎠

since

cv = --s-
X

s = ( cv ) ( X )

s = ( 0.12 ) ( 0.07 )

s = 0.008

A-4 Applied Biosystems StepOne™ and StepOnePlus™ Real-Time PCR Systems Reagent Guide
 Appendix A Formulas

Standard Deviation Calculation Using the Comparative

Method
Example The CT data used to determine the amounts of c-myc and GAPDH mRNA shown in
the table on page A-2 are used to illustrate the ΔΔCT calculation. The table below
shows the average CT results for the human brain, kidney, liver, and lung samples and
how these CTs are manipulated to determine ΔCT, ΔΔCT, and the relative amount of c-
myc mRNA. The results are comparable to the relative c-myc levels determined
using the standard curve method.

c-myc GAPDH ΔCT ΔΔCT c-mycN

Tissue
Average CT Average CT c-myc–GAPDH ‡ ΔCT–ΔCT, Brain § Rel. to Brain #

Brain 30.49±0.15 23.63±0.09 6.86±0.17 0.00±0.17 1.0

(0.9–1.1)

Kidney 27.03±0.06 22.66±0.08 4.37±0.10 –2.50±0.10 5.6

(5.3–6.0)

Liver 26.25±0.07 24.60±0.07 1.65±0.10 –5.21±0.10 37.0

(34.5–39.7)

Lung 25.83±0.07 23.01±0.07 2.81±0.10 –4.05±0.10 16.5

(15.4–17.7)

‡ The ΔCT value is determined by subtracting the average GAPDH CT value from the average c-myc CT value. The standard
deviation of the difference is calculated from the standard deviations of the c-myc and GAPDH values. See “Formula”
on page A-6.
§ The calculation of ΔΔCT involves subtraction by the ΔCT reference sample value. This is subtraction of an arbitrary
constant, so the standard deviation of ΔΔCT is the same as the standard deviation of the ΔCT value.
# The range given for c-mycN relative to brain is determined by evaluating the expression: 2 –ΔΔCT
with ΔΔCT + s and ΔΔCT – s, where s = the standard deviation of the ΔΔCT value.

Applied Biosystems StepOne™ and StepOnePlus™ Real-Time PCR Systems Reagent Guide A-5
Appendix A Formulas

Formula The ΔCΤ value is determined by subtracting the average GAPDH CT value from the
average c-myc CT value. The standard deviation of the difference is calculated from
the standard deviations of the c-myc and GAPDH values using the following
formula:

2 2
s = s1 + s2

where:
s = std dev
Using the brain sample from the table on page A-5 as an example:

s 1 = 0.15

and

s 2 = 0.09

2 2
s= ( 0.15 ) + ( 0.09 ) = 0.17

A-6 Applied Biosystems StepOne™ and StepOnePlus™ Real-Time PCR Systems Reagent Guide
 Appendix A Formulas

Formula for Comparative CT (ΔΔCT) Experiments

Formula The quantity of target, normalized to an endogenous control and relative to a
reference sample, is calculated by:
2 –ΔΔCT

Derivation of the The equation that describes the exponential amplification of PCR is:
Formula
Xn = Xo × ( 1 + EX ) n

where:
• xn = number of target molecules at cycle n
• xo = initial number of target molecules
• Ex = efficiency of target amplification
• n = number of cycles
• Xo =initial number of target molecules
The threshold cycle (CT) indicates the fractional cycle number at which the quantity
of amplified target reaches a specified threshold. Thus,

X T = X o × ( 1 + E X ) C T, X = K X

where:
• xT = threshold number of target molecules
• CT, X = threshold cycle for target amplification
• KX = constant
A similar equation for the endogenous control reaction is:

R T = R o × ( 1 + E R ) C T, R = K R

where:
• RT = threshold number of reference molecules
• Ro = initial number of reference molecules
• ER = efficiency of reference amplification
• CT, R = threshold cycle for reference amplification
• KR = constant
Dividing XT by RT yields the following expression:

C
XT X o × ( 1 + E X ) T, X K X
------ = ---------------------------------------
C
= ------ = K
RT R o × ( 1 + E R ) T, R K R

Applied Biosystems StepOne™ and StepOnePlus™ Real-Time PCR Systems Reagent Guide A-7
Appendix A Formulas

The exact values of XT and RT depend on a number of factors, including:

• Reporter dye used in the probe
• Sequence context effects on the fluorescence properties of the probe
• Efficiency of probe cleavage
• Purity of the probe
• Setting of the fluorescence threshold.
Therefore, the constant K does not have to be equal to 1.
Assuming efficiencies of the target and the reference are the same:
EX = E R = E

Xo C –C
----- × ( 1 + E ) T, X T, R= K
Ro

Or

ΔC T
XN × ( 1 + E ) =K

where:
• XN = XO/RO, the normalized quantity of target
• ΔCT = CT, X – CT, R, the difference in threshold cycles for target and reference
Rearranging gives the following expression:

– ΔC T
XN = K × ( 1 + E )

The final step is to divide the XN for any sample (q) by the XN for the reference
sample (cb):

X N, q – ΔC
K × ( 1 + E ) T, q – ΔΔC T
----------- = ----------------------------------------
Δ C
- == ( 1 + E )
X N, cb K × (1 + E )
– T , c b

where:
• ΔΔCT = ΔCT, q – ΔCT, cb
For amplicons designed and optimized according to Applied Biosystems Assay
Design Guidelines (amplicon size <150 bp), the efficiency is close to 1. Therefore,
the quantity of target, normalized to an endogenous control and relative to a
reference sample, is given by:
2 –ΔΔCT

A-8 Applied Biosystems StepOne™ and StepOnePlus™ Real-Time PCR Systems Reagent Guide
Primer Limiting in Multiplex PCR B B
To generate an accurate multiplex assay, it is important that the amplification of one
species does not dominate amplification of the other. Otherwise, the amplification of
a highly abundant species can prevent the less-abundant species from amplifying
efficiently. If the less-abundant species does not amplify efficiently, your experiment
may produce inaccurate results. Or, in severe cases, detection of the less-abundant
species may be inhibited completely. You can avoid this situation by limiting the
concentrations of the primers used to amplify the more abundant species, thereby
“turning off ” the amplification soon after the CT has been established.
Primer limitation results in the reaction components common to both assays not
being exhausted, allowing the amplification of the less-abundant species to continue
at high efficiency. If the more abundant species is not known, you should determine
it before performing multiplex PCR by running both targets in separate tubes. Both
amplifications should be primer limited if neither species is consistently more
abundant.

Considering In applying the primer limitation to target and endogenous control amplifications, the
Relative relative abundance of the two species must be considered. For quantitation
Abundance of the experiments, it is possible to use rRNA as an endogenous control. The concentration
Target and of rRNA in total RNA is always greater than the concentration of any target mRNA.
Reference Therefore, in multiplex reactions amplifying both target and rRNA, only the
concentrations of the rRNA primers need to be limited.

Limiting Primer To define limiting primer concentrations, run a matrix of forward and reverse primer
Matrix concentrations using the value of the minimum initial template. The goal is to
identify primer concentrations that reduce the ΔRn value of the assay without
affecting the CT value. The table below illustrates a recommended matrix of forward
and reverse primers varying in concentration from 20 to 100 nM.
Note: Although following all design criteria does facilitate the ability to identify
limiting primer concentrations, it may not be possible for all assays. If a limiting
primer matrix experiment does not enable the identification of primer limiting
concentrations, it will be necessary to redesign at least one primer or run the
reactions in separate tubes.

Forward: 100 nM 100 nM 100 nM 100 nM 100 nM

Reverse: 100 nM 80 nM 60 nM 40 nM 20 nM

Forward: 80 nM 80 nM 80 nM 80 nM 80 nM
Reverse: 100 nM 80 nM 60 nM 40 nM 20 nM

Forward: 60 nM 60 nM 60 nM 60 nM 60 nM
Reverse: 100 nM 80 nM 60 nM 40 nM 20 nM

Applied Biosystems StepOne™ and StepOnePlus™ Real-Time PCR Systems Reagent Guide B-1
Appendix B Primer Limiting in Multiplex PCR

Forward: 100 nM 100 nM 100 nM 100 nM 100 nM

Reverse: 100 nM 80 nM 60 nM 40 nM 20 nM

Forward: 40 nM 40 nM 40 nM 40 nM 40 nM
Reverse: 100 nM 80 nM 60 nM 40 nM 20 nM

Forward: 20 nM 20 nM 20 nM 20 nM 20 nM
Reverse: 100 nM 80 nM 60 nM 40 nM 20 nM

Example The results of a limiting primer matrix experiment are shown in Figure B-1 on
page B-3:
• Figure B-1a shows that only when lowering the primer concentrations below
approximately 50 nM is the CT value significantly affected. The plateau area
shows the region in which suitable primer limiting concentrations can be found.
In this area, the CT (and therefore the corresponding quantitation value) is
unchanged, whereas the ΔRn value and corresponding product yield are
significantly reduced.
• Figure B-1b shows the corresponding relationship between primer
concentrations and ΔRn. The figure demonstrates that lower product yields can
be achieved by decreasing forward and reverse primer concentrations.
For this example, an appropriate selection of primer limiting concentrations would
be at least 50 nM forward and reverse primer. Probe concentration should be kept at
an optimal level even when an assay is primer limited to ensure that the signal
produced is large enough for accurate multicomponenting by the software.

B-2 Applied Biosystems StepOne™ and StepOnePlus™ Real-Time PCR Systems Reagent Guide
 Appendix B Primer Limiting in Multiplex PCR

Figure B-1 Results from Limiting Primer Matrix experiment

(a) Shows how CT value is affected by variation in forward and reverse primer
concentrations. Plateau region indicated shows area where CT value remains
constant.
(b) Shows reduction in ΔRn values as primer concentration decreases.

Applied Biosystems StepOne™ and StepOnePlus™ Real-Time PCR Systems Reagent Guide B-3
Appendix B Primer Limiting in Multiplex PCR

B-4 Applied Biosystems StepOne™ and StepOnePlus™ Real-Time PCR Systems Reagent Guide
Assay Design Guidelines C C
About Assay If you are designing your own assays (primers and probes), Applied Biosystems
Design recommends that you follow the Applied Biosystems Assay Design Guidelines. The
Guidelines Assay Design Guidelines specify that you:
1. Design primers and probes using Primer Express® Software – The Primer
Express software uses a set of default parameters to automatically select primer
and probe sets.
2. Select the appropriate reagents – There are several TaqMan® and SYBR®
Green reagents available. The reagents you use depend on your assay type.
3. Use the recommended thermal cycling conditions – Use the thermal cycling
conditions recommended for your sample (DNA/cDNA, RNA for 1-step PCR,
and RNA for 2-step PCR).
Note: Thermal cycling conditions for Fast reagents differ from thermal cycling
conditions for standard reagents.
4. Use default primer and probe concentrations or optimize primer and probe
concentrations – When you use Applied Biosystems Assay Design Guidelines,
you can use default primer and probe concentrations for non-multiplex
optimized assays, or you can optimize primer and probe concentrations.
IMPORTANT! These steps provide a rapid and reliable system for assay design and
optimization only when used in their entirety. Adopt the system as a whole to
achieve the highest level of success.
Note: Applied Biosystems Assay Design Guidelines do not guarantee that all assays
will provide the same level of performance and sensitivity. Even the most scrupulous
design parameters cannot account for all the possible variations between two
different assay systems.

Conclusions for In general, the following conclusions can be made when you use the Assay Design
Quantitation Guidelines for quantitation experiments:
Experiments • For most TaqMan assays, a concentration of 900-nM primers and 250-nM probe
results in a highly reproducible and sensitive assay when using DNA or cDNA
as a template.
• Due to the nonspecific nature of its detection, SYBR® Green I dye primer
optimization should be bypassed only with caution. However, if all guidelines
are followed, concentrations of 50-nM forward and reverse primers generally
provide robust amplification with a good level of specificity when using DNA
or cDNA as a template. Verify this assumption by checking for nonspecific
product formation with either melt curve or gel analysis.
• Most TaqMan assays should enable detection and accurate quantitation to
<50 copies of a target, with even greater sensitivity possible.

Applied Biosystems StepOne™ and StepOnePlus™ Real-Time PCR Systems Reagent Guide C-1
Appendix C Assay Design Guidelines

• SYBR Green assays are capable of similar performance; however, nonspecific

product formation can potentially increase the minimum detection limit.

Conclusions for In general, the following conclusion can be made when you use the Assay Design
Genotyping Guidelines for genotyping experiments:
Experiments
You can use 900-nM primers, a 200-nM probe, and 1 to 20 ng of genomic DNA to
achieve reproducible and sensitive assay results.

C-2 Applied Biosystems StepOne™ and StepOnePlus™ Real-Time PCR Systems Reagent Guide
Reagent Part Numbers D D
This appendix covers:
Quantitation Experiments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . D-2
Genotyping Experiments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . D-5
Presence/Absence Experiments. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . D-7

Applied Biosystems StepOne™ and StepOnePlus™ Real-Time PCR Systems Reagent Guide D-1
Appendix D Reagent Part Numbers

Quantitation Experiments

Inventoried/Made to Order Assays

Assays
Product Part Number

TaqMan® Gene Expression Assays For information on the latest available

products and specific product uses,
TaqMan® Endogenous Control Assays refer to the Applied Biosystems Web
site:
Note: FAM™-dye labeled TaqMan® Endogenous
Control Assays are included as Inventoried http://www.appliedbiosystems.com/
TaqMan Gene Expression Assays.

Custom TaqMan® Gene Expression Assays

Master Mixes
Master Mix Part Number

TaqMan® Gene Expression Master Mix, 1-Pack (1 × 5 mL), 4369016

200 reactions

TaqMan® Gene Expression Master Mix, 10-Pack (10 × 5 mL), 4369542

2000 reactions

TaqMan® Fast Universal PCR Master Mix (2✕), No AmpErase® UNG, 4352042
250 Reactions

TaqMan® 2✕ Universal PCR Master Mix, 200 Reactions 4304437

TaqMan® 2✕ Universal PCR Master Mix, 2000 Reactions 4326708

10-Pack, TaqMan® 2✕ Universal PCR Master Mix 4305719

TaqMan® 2✕ Universal PCR Master Mix, No AmpErase® UNG, 200 4324018

Reactions

TaqMan® 2✕ Universal PCR Master Mix, No AmpErase® UNG, 2000 4326614

Reactions

10-Pack, TaqMan® 2✕ Universal PCR Master Mix, No AmpErase® UNG 4324020

Custom Assays

Assays
Product Part Number

Custom TaqMan® Probes and Primers For information on the latest available
products and specific product uses,
refer to the Applied Biosystems Web
site:
http://www.appliedbiosystems.com/

D-2 Applied Biosystems StepOne™ and StepOnePlus™ Real-Time PCR Systems Reagent Guide
 Appendix D Reagent Part Numbers

DNA or cDNA
Reagent Kit Part Number
Quantitation
TaqMan® reagents TaqMan® Gene Expression Master Mix, 1-Pack 4369016
(1 × 5 mL), 200 reactions

TaqMan® Gene Expression Master Mix, 10-Pack 4369542

(10 × 5 mL), 2000 reactions

TaqMan® Fast Universal PCR Master Mix (2✕), No 4352042

AmpErase® UNG, 250 Reactions

TaqMan® 2✕ Universal PCR Master Mix, 200 4304437

Reactions

TaqMan® 2✕ Universal PCR Master Mix, 2000 4326708

Reactions

10-Pack, TaqMan® 2✕ Universal PCR Master Mix 4305719

TaqMan® 2✕ Universal PCR Master Mix, No 4324018

AmpErase® UNG, 200 Reactions

TaqMan® 2✕ Universal PCR Master Mix, No 4326614

AmpErase® UNG, 2000 Reactions

10-Pack, TaqMan® 2✕ Universal PCR Master Mix, 4324020

No AmpErase® UNG

TaqMan® PCR Core Reagents Kit, 200 Reactions N808-0228

SYBR® Green Power SYBR® Green PCR Master Mix (1 mL), 4368577
reagents 40 reactions

Power SYBR® Green PCR Master Mix (5-mL), 4367659

200 reactions

Power SYBR® Green PCR Master Mix 4368708

(10 × 5-mL), 2000 reactions

Power SYBR® Green PCR Master Mix (50 mL), 4367660

2000 reactions

SYBR® Green PCR Master Mix (1-mL), 4344463

40 reactions

SYBR® Green PCR Master Mix (5-mL), 4309155

200 reactions

SYBR® Green PCR Master Mix (50-mL), 4334973

2000 reactions

SYBR® Green PCR Core Reagents, 200 Reactions 4304886

Applied Biosystems StepOne™ and StepOnePlus™ Real-Time PCR Systems Reagent Guide D-3
Appendix D Reagent Part Numbers

RNA Quantitation
Reagent Kit Part Number
Using 1-Step
RT-PCR TaqMan® reagents TaqMan® One-Step RT-PCR Master Mix 4309169
Reagents Kit

TaqMan® EZ RT-PCR Core Reagents N808-0236

Note: Use the TaqMan® EZ RT-PCR Core
Reagents when a high-temperature RT step is
required.

SYBR® Green Power SYBR® Green RT-PCR Reagents Kit 4368711

reagents
SYBR® Green RT-PCR Reagents 4310179

RNA Quantitation
Reagent Step Kit Part Number
Using 2-Step
RT-PCR TaqMan® PCR step only TaqMan® Gene Expression Master 4369016
reagents Mix, 1-Pack (1 × 5 mL),
200 reactions

TaqMan® Gene Expression Master 4369542

Mix, 10-Pack (10 × 5 mL),
2000 reactions

TaqMan® 2✕ Universal PCR Master 4304437

Mix

TaqMan® Fast Universal PCR Master 4352042

Mix (2✕), No AmpErase® UNG

RT step only High-Capacity cDNA Reverse 4374966

Transcription Kit

TaqMan® Reverse Transcription N808-234

Reagents

Both RT and TaqMan® Gold RT PCR kit N808-232

PCR steps

SYBR® Green PCR step only Power SYBR® Green PCR Master 4367659
reagents Mix

SYBR® Green PCR Master Mix 4309155

RT step only High-Capacity cDNA Reverse 4374966

Transcription Kit

TaqMan® Reverse Transcription N808-234

Reagents

Both RT and Power SYBR® Green RT-PCR 4368711

PCR steps Reagents Kit

SYBR® Green RT-PCR Reagents 4310179

D-4 Applied Biosystems StepOne™ and StepOnePlus™ Real-Time PCR Systems Reagent Guide
 Appendix D Reagent Part Numbers

Genotyping Experiments

Pre-Designed/Validated Assays

Assays
Product Part Number

TaqMan® SNP Genotyping Assays For information on the latest available

products and specific product uses,
TaqMan® Drug Metabolism Genotyping Assays refer to the Applied Biosystems Web
site:
Pre-Developed TaqMan® Assay Reagents for
Allelic Discrimination http://www.appliedbiosystems.com/

Master Mixes
Master Mix Part Number

TaqMan® Genotyping Master Mix, 1-Pack (1 × 10 mL), 400 reactions 4371355

TaqMan® Genotyping Master Mix, 1 Bulk Pack (1 × 50 mL), 4371357

2000 reactions

TaqMan® 2✕ Universal PCR Master Mix, 200 Reactions 4304437

TaqMan® 2✕ Universal PCR Master Mix, 2000 Reactions 4326708

10-Pack, TaqMan® 2✕ Universal PCR Master Mix 4305719

TaqMan® 2✕ Universal PCR Master Mix, No AmpErase® UNG, 200 4324018

Reactions

TaqMan® 2✕ Universal PCR Master Mix, No AmpErase® UNG, 2000 4326614

Reactions

10-Pack, TaqMan® 2✕ Universal PCR Master Mix, No AmpErase® UNG 4324020

Note: Genotyping experiments are not supported using Fast or SYBR® Green
master mixes and protocols.

Custom Assays

Assays
Product Part Number

Custom TaqMan® SNP Genotyping Assays For information on the latest available
products and specific product uses,
refer to the Applied Biosystems Web
site:
http://www.appliedbiosystems.com/

Applied Biosystems StepOne™ and StepOnePlus™ Real-Time PCR Systems Reagent Guide D-5
Appendix D Reagent Part Numbers

Master Mixes
Master Mix Part Number

TaqMan® Genotyping Master Mix, 1-Pack (1 × 10 mL), 400 reactions 4371355

TaqMan® Genotyping Master Mix, 1 Bulk Pack (1 × 50 mL), 4371357

2000 reactions

TaqMan® 2✕ Universal PCR Master Mix, 200 Reactions 4304437

TaqMan® 2✕ Universal PCR Master Mix, 2000 Reactions 4326708

10-Pack, TaqMan® 2✕ Universal PCR Master Mix 4305719

TaqMan® 2✕ Universal PCR Master Mix, No AmpErase® UNG, 200 4324018

Reactions

TaqMan® 2✕ Universal PCR Master Mix, No AmpErase® UNG, 2000 4326614

Reactions

10-Pack, TaqMan® 2✕ Universal PCR Master Mix, No AmpErase® UNG 4324020

Note: Genotyping experiments are not supported using Fast or SYBR® Green
master mixes and protocols.

D-6 Applied Biosystems StepOne™ and StepOnePlus™ Real-Time PCR Systems Reagent Guide
 Appendix D Reagent Part Numbers

Presence/Absence Experiments

Inventoried/Made to Order Assays

Assays
Product Part Number

TaqMan® Gene Expression Assays For information on the latest available

products and specific product uses,
TaqMan® Endogenous Control Assays refer to the Applied Biosystems Web
site:
Note: FAM™-dye labeled TaqMan® Endogenous
Control Assays are included as Inventoried http://www.appliedbiosystems.com/
TaqMan Gene Expression Assays.

Custom TaqMan® Gene Expression Assays

TaqMan
Kits Part Number
Exogenous IPC
Reagents TaqMan® Exogenous Internal Positive Control Reagents with 4308320
TaqMan® 2✕ Universal PCR Master Mix (with VIC® dye)

TaqMan® Exogenous Internal Positive Control Reagents 4308323

Note: If you are using this kit, you will need to purchase one of these
TaqMan® reagents separately:
• TaqMan® 2✕ Universal PCR Master Mix (PN 4304437)
• TaqMan® PCR Core Reagents Kit (PN N808-0228)

IMPORTANT! The kits listed above contain TAMRA™ dye-labeled probes; Applied
Biosystems does not recommend the use of TAMRA dye as a reporter or quencher
with the StepOne system. The kits may be used with the StepOnePlus system;
TAMRA dye may be used as a reporter or quencher with the StepOnePlus system.

Applied Biosystems StepOne™ and StepOnePlus™ Real-Time PCR Systems Reagent Guide D-7
Appendix D Reagent Part Numbers

Master Mixes
Master Mix Part Number

TaqMan® Gene Expression Master Mix, 1-Pack (1 × 5 mL), 4369016

200 reactions

TaqMan® Gene Expression Master Mix, 10-Pack (10 × 5 mL), 4369542

2000 reactions

TaqMan® 2✕ Universal PCR Master Mix, 200 Reactions 4304437

TaqMan® 2✕ Universal PCR Master Mix, 2000 Reactions 4326708

10-Pack, TaqMan® 2✕ Universal PCR Master Mix 4305719

TaqMan® 2✕ Universal PCR Master Mix, No AmpErase® UNG, 200 4324018

Reactions

TaqMan® 2✕ Universal PCR Master Mix, No AmpErase® UNG, 2000 4326614

Reactions

10-Pack, TaqMan® 2✕ Universal PCR Master Mix, No AmpErase® UNG 4324020

TaqMan® PCR Core Reagents Kit N808-0228

Note: If you purchase the TaqMan® Exogenous Internal Positive Control Reagents
with TaqMan® 2✕ Universal PCR Master Mix kit (PN 4308320), you do not need to
purchase the master mix separately.
Note: Presence/absence experiments are not supported using Fast or SYBR® Green
master mixes and protocols.

Custom Assays

Assays
Product Part Number

Custom TaqMan® Probes and Primers For information on the latest available
products and specific product uses,
refer to the Applied Biosystems Web
site:
http://www.appliedbiosystems.com/

D-8 Applied Biosystems StepOne™ and StepOnePlus™ Real-Time PCR Systems Reagent Guide
 Appendix D Reagent Part Numbers

DNA or cDNA
Kit Part Number
Quantitation
TaqMan® Gene Expression Master Mix, 1-Pack (1 × 5 mL), 200 reactions 4369016

TaqMan® Gene Expression Master Mix, 10-Pack (10 × 5 mL), 4369542

2000 reactions

TaqMan® 2✕ Universal PCR Master Mix, 200 Reactions 4304437

TaqMan® 2✕ Universal PCR Master Mix, 2000 Reactions 4326708

10-Pack, TaqMan® 2✕ Universal PCR Master Mix 4305719

TaqMan® 2✕ Universal PCR Master Mix, No AmpErase® UNG, 200 4324018

Reactions

TaqMan® 2✕ Universal PCR Master Mix, No AmpErase® UNG, 2000 4326614

Reactions

10-Pack, TaqMan® 2✕ Universal PCR Master Mix, No AmpErase® UNG 4324020

TaqMan® PCR Core Reagents Kit, 200 Reactions N808-0228

Note: Presence/absence experiments are not supported using Fast or SYBR® Green
master mixes and protocols.

RNA Quantitation
Kit Part Number
Using 1-Step
RT-PCR TaqMan® One-Step RT-PCR Master Mix Reagents Kit 4309169

TaqMan® EZ RT-PCR Core Reagents N808-0236

Note: Use the TaqMan® EZ RT-PCR Core Reagents when a high-
temperature RT step is required.

Note: Presence/absence experiments are not supported using Fast or SYBR® Green
master mixes and protocols.

RNA Quantitation
Step Kit Part Number
Using 2-Step
RT-PCR PCR step only TaqMan® Gene Expression Master Mix, 4369016
1-Pack (1 × 5 mL), 200 reactions

TaqMan® Gene Expression Master Mix, 4369542

10-Pack (10 × 5 mL), 2000 reactions

TaqMan® 2✕ Universal PCR Master Mix 4304437

RT step only High-Capacity cDNA Reverse Transcription 4374966

Kit

TaqMan® Reverse Transcription Reagents N808-234

Both RT and PCR steps TaqMan® Gold RT PCR kit N808-232

Note: Presence/absence experiments are not supported using Fast or SYBR® Green
master mixes and protocols.

Applied Biosystems StepOne™ and StepOnePlus™ Real-Time PCR Systems Reagent Guide D-9
Appendix D Reagent Part Numbers

D-10 Applied Biosystems StepOne™ and StepOnePlus™ Real-Time PCR Systems Reagent Guide
Bibliography

Afonina, I., Zivarts, M., Kutyavin, I., et al. 1997. Efficient priming of PCR with
short oligonucleotides conjugated to a minor groove binder. Nucleic Acids Res.
25:2657–2660.
Förster, V. T. 1948. Zwischenmolekulare Energiewanderung und Fluoreszenz. Ann.
Physics (Leipzig) 2:55–75.
Higuchi, R., Dollinger, G., Walsh, P.S., and Griffith, R. 1992. Simultaneous
amplification and detection of specific DNA sequences. Biotechnology 10:413–417.
Higuchi, R., Fockler, C., Dollinger, G., and Watson, R. 1993. Kinetic PCR:Real time
monitoring of DNA amplification reactions. Biotechnology 11:1026–1030.
Kutyavin, I.V., Lukhtanov, E.A., Gamper, H.B., and Meyer, R.B. 1997.
Oligonucleotides with conjugated dihydropyrroloindole tripeptides: base
composition and backbone effects on hybridization. Nucleic Acids Res.
25:3718–3723.
Kwok, S. and Higuchi, R. 1989. Avoiding false positives with PCR. Nature
339:237–238.
Livak, K.J., and Schmittgen, T.D. 2001. Analysis of Relative Gene Expression Data
Using Real-Time Quantitative PCR and the 2–ΔΔCT Method. Methods 25:402–408.
Longo, M.C., Berninger, M.S., and Hartley, J.L. 1990. Use of uracil DNA
glycosylase to control carry-over contamination in polymerase chain reactions. Gene
93:125–128.
Saiki, R.K., Scharf, S., Faloona, F., et al. 1985. Enzymatic amplification of β-globin
genomic sequences and restriction site analysis for diagnosis of sickle cell anemia.
Science 230:1350–1354.

Applied Biosystems StepOne™ and StepOnePlus™ Real-Time PCR Systems Reagent Guide Bibliography-1
Bibliography

Bibliography-2 Applied Biosystems StepOne™ and StepOnePlus™ Real-Time PCR Systems Reagent Guide
Glossary

Advanced Setup In the StepOne™ software, a feature that allows you to set up your experiment
according to your experiment design. Advanced Setup provides you with maximum
flexibility in the design and setup of your experiment.

AIF See assay information file (AIF).

allele For a given target, any of the different sequences that occurs in the population.

allelic discrimina- Display of data collected during the post-PCR read. The allelic discrimination plot is
tion plot a graph of the normalized reporter signal from the allele 1 probe plotted against the
normalized reporter signal from the allele 2 probe.

amplicon A segment of DNA amplified during PCR.

amplification Part of the instrument run in which PCR produces amplification of the target. For
quantitation experiments, fluorescence data collected during amplification are
displayed in an amplification plot, and the data are used to calculate results. For
genotyping or presence/absence experiments, fluorescence data collected during
amplification are displayed in an amplification plot, and the data can be used for
troubleshooting.

amplification Calculation of efficiency of the PCR amplification. The amplification efficiency is

efficiency (EFF%) calculated using the slope of the regression line in the standard curve. A slope close to
−3.32 indicates optimal, 100% PCR amplification efficiency. Factors that affect
amplification efficiency:
• Range of standard quantities – To increase the accuracy and precision of the
efficiency measurement, use a broad range of standard quantities, 5 to 6 logs
(105 to 106 fold).
• Number of standard replicates – To increase the precision of the standard
quantities and decrease the effects of pipetting inaccuracies, include replicates.
• PCR inhibitors – PCR inhibitors in the reaction can reduce amplification and
alter measurements of the efficiency.

amplification plot Display of data collected during the cycling stage of PCR amplification. Can be
viewed as:
• Baseline-corrected normalized reporter (ΔRn) vs. cycle
• Normalized reporter (Rn) vs. cycle
• Threshold cycle (CT) vs. well

Applied Biosystems StepOne™ and StepOnePlus™ Real-Time PCR Systems Reagent Guide Glossary-1
Glossary

amplification stage Part of the instrument run in which PCR produces amplification of the target. The
amplification stage is called a cycling stage in the thermal profile and consists of
denaturing, primer annealing, and polymerization steps that are repeated.
For quantitation experiments, fluorescence data collected during the amplification
stage are displayed in an amplification plot, and the data are used to calculate results.
For genotyping or presence/absence experiments, fluorescence data collected during
the amplification stage are displayed in an amplification plot, and the data can be used
for troubleshooting. See also cycling stage.

assay In the StepOne™ and StepOnePlus™ systems, a PCR reaction mix that contains
primers to amplify a target and a reagent to detect the amplified target.

Assay ID Identifier assigned by Applied Biosystems to TaqMan® Gene Expression Assays and
TaqMan® SNP Genotyping Assays.

assay information Data file on a CD shipped with each assay order. The file name includes the number
file (AIF) from the barcode on the plate. The information in the AIF is provided in a tab-
delimited format.

assay mix PCR reaction component in Applied Biosystems TaqMan® Gene Expression Assays
and TaqMan® SNP Genotyping Assays. The assay mix contains primers designed to
amplify a target and a TaqMan® probe designed to detect amplification of the target.

AutoDelta In the run method, a setting to increase or decrease the temperature and/or time for a
step with each subsequent cycle in a cycling stage. When AutoDelta is enabled for a
cycling stage, the settings are indicated by an icon in the thermal profile:
• AutoDelta on:
• AutoDelta off:

automatic baseline An analysis setting in which the software calculates the baseline start and end values
for the amplification plot. You can apply the automatic baseline setting to specific
wells in the reaction plate. See also baseline.

automatic CT An analysis setting in which the software calculates the baseline start and end values
and the threshold in the amplification plot. The software uses the baseline and
threshold to calculate the threshold cycle (CT). See also threshold cycle (CT).

baseline In the amplification plot, a line fit to the fluorescence levels during the initial stages
of PCR, when there is little change in fluorescence signal.

Glossary-2 Applied Biosystems StepOne™ and StepOnePlus™ Real-Time PCR Systems Reagent Guide
 Glossary

baseline-corrected The magnitude of normalized fluorescence signal generated by the reporter:

normalized reporter
(ΔRn) 1. In experiments that contain data from real-time PCR, the magnitude of
normalized fluorescence signal generated by the reporter at each cycle during
the PCR amplification. In the ΔRn vs. Cycle amplification plot, ΔRn is
calculated at each cycle as:
ΔRn (cycle) = Rn (cycle) − Rn (baseline), where Rn = normalized reporter

2. In genotyping experiments and presence/absence experiments, the difference in

normalized fluorescence signal generated by the reporter between the pre-PCR
read and the post-PCR read. In the allelic discrimination plot (genotyping
experiments) and the presence/absence plot (presence/absence experiments),
ΔRn is calculated as:
ΔRn = Rn (post-PCR read) − Rn (pre-PCR read), where Rn = normalized
reporter
See also normalized reporter (Rn).

blocked IPC In presence/absence experiments, a reaction that contains IPC blocking agent, which
blocks amplification of the internal positive control (IPC). In the StepOne™ software,
the task for the IPC target in wells that contain IPC blocking agent. See also negative
control-blocked IPC wells.

calibrator See reference sample.

chemistry See reagents.

colocated layout A system layout in which the StepOne™ or StepOnePlus™ instrument is directly
connected to a colocated computer by the yellow cable. In this layout, you can control
the instrument with the StepOne™ software on the colocated computer or with the
instrument touchscreen.

comparative CT Method for determining relative target quantity in samples. With the comparative CT
(ΔΔCT) method (ΔΔCT) method, the StepOne™ software measures amplification of the target and of the
endogenous control in samples and in a reference sample. Measurements are
normalized using the endogenous control. The software determines the relative
quantity of target in each sample by comparing normalized target quantity in each
sample to normalized target quantity in the reference sample.

CT See threshold cycle (CT).

custom dye Dye that is not supplied by Applied Biosystems. Custom dyes may be adapted for use
in experiments on the StepOne™ and StepOnePlus™ systems. When using custom
dyes, the custom dye should be added to the Dye Library and a custom dye calibration
performed.
IMPORTANT! Applied Biosystems does not recommend the use of TAMRA™ dye as
reporter or quencher with the StepOne™ system. TAMRA dye may be used as a
reporter or quencher with the StepOnePlus™ system.

cycle threshold See threshold cycle (CT).

Applied Biosystems StepOne™ and StepOnePlus™ Real-Time PCR Systems Reagent Guide Glossary-3
Glossary

cycling stage In the thermal profile, a stage that is repeated. A cycling stage is also called an
amplification stage. For cycling stages, you can enable AutoDelta settings. See also
amplification stage.

data collection A process during the instrument run in which an instrument component detects
fluorescence data from each well of the reaction plate. The instrument transforms the
signal to electronic data, and the data are saved in the experiment file. In the
StepOne™ software, a data collection point is indicated by an icon in the thermal
profile:
• Data collection on:
• Data collection off:

delta Rn (ΔRn) See baseline-corrected normalized reporter (ΔRn).

derivative reporter The negative first-derivative of the normalized fluorescence generated by the reporter
(−Rn′) during PCR amplification. In the derivative reporter (–Rn′) vs. temperature melt
curve, the derivative reporter signal is displayed in the y-axis.

Design Wizard A feature in the StepOne™ software that helps you set up your experiment by guiding
you through best practices as you enter your experiment design.

diluent A reagent used to dilute a sample or standard before adding it to the PCR reaction. The
diluent can be water or buffer.

Diluted Sample In the StepOne™ software, a field displayed on the Sample Dilution Calculations tab
Concentration (10✕ of the Reaction Setup screen. For this field, enter the sample concentration you want
for Reaction Mix) to use to add to the reaction mix for all samples in the experiment. “10✕ for Reaction
Mix” indicates that the software assumes the sample or standard component of the
reaction mix is at a 10✕ concentration. For example, if the diluted sample
concentration is 50.0 ng/μL (10✕), the final sample concentration in the reaction is 5
ng/μL (1✕).

dilution factor See serial factor.

dissociation curve See melt curve.

EFF% See amplification efficiency (EFF%).

endogenous A target or gene that should be expressed at similar levels in all samples you are
control testing. Endogenous controls are used in relative standard curve and comparative CT
(ΔΔCT) experiments to normalize fluorescence signals for the target you are
quantifying. Housekeeping genes can be used as endogenous controls. See also
housekeeping gene.

endpoint read See post-PCR read.

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 Glossary

experiment Refers to the entire process of performing a run using the StepOne™ or StepOnePlus™
systems, including setup, run, and analysis. The types of experiments you can perform
using the StepOne and StepOnePlus systems:
• Quantitation - standard curve
• Quantitation - relative standard curve
• Quantitation - comparative CT (ΔΔCT)
• Melt curve
• Genotyping
• Presence/absence

experiment name Entered during experiment setup, the name that is used to identify the experiment.
Experiment names cannot exceed 100 characters and cannot include any of the
following characters: forward slash (/), backslash (\), greater than sign (>), less than
sign (<), asterisk (*), question mark (?), quotation mark ("), vertical line (|), colon (:),
or semicolon (;).

experiment type The type of experiment you are performing using the StepOne™ or StepOnePlus™
system:
• Standard curve
• Comparative CT (ΔΔCT)
• Relative standard curve
• Melt curve (not available in the Design Wizard)
• Genotyping
• Presence/absence
The experiment type you select affects the setup, run, and analysis.

forward primer Oligonucleotide that flanks the 5′ end of the amplicon. The reverse primer and the
forward primer are used together in PCR reactions to amplify the target.

holding stage In the thermal profile, a stage that includes one or more steps. You can add a holding
stage to the thermal profile to activate enzymes, to inactivate enzymes, or to incubate
a reaction.

housekeeping gene A gene that is involved in basic cellular functions and is constitutively expressed.
Housekeeping genes can be used as endogenous controls. See also endogenous
control.

internal positive In presence/absence experiments, a short synthetic DNA template that is added to
control (IPC) PCR reactions. You can use the IPC to distinguish between true negative results (that
is, the target is absent in the samples) and negative results caused by PCR inhibitors,
incorrect assay setup, or reagent or instrument failure.

inventoried assays TaqMan® Gene Expression Assays and TaqMan® SNP Genotyping Assays that have
been previously manufactured, passed quality control specifications, and stored in
inventory.

Applied Biosystems StepOne™ and StepOnePlus™ Real-Time PCR Systems Reagent Guide Glossary-5
Glossary

IPC In presence/absence experiments, abbreviation for internal positive control (IPC). In

the StepOne™ software, the task for the IPC target in wells that contain the IPC and
do not contain IPC blocking agent. See also internal positive control (IPC).

IPC blocking agent Reagent added to PCR reactions to block amplification of the internal positive control
(IPC).

IPC+ See negative control-IPC wells.

made-to-order TaqMan® Gene Expression Assays or TaqMan® SNP Genotyping Assays that are
assays manufactured at the time of order. Only assays that pass manufacturing quality control
specifications are shipped.

manual baseline An analysis setting in which you enter the baseline start and end values for the
amplification plot. You can apply the manual baseline setting to specific wells in the
reaction plate.

manual CT An analysis setting in which you enter the threshold value and select whether to use
automatic baseline or manual baseline values. The software uses the baseline and the
threshold values to calculate the threshold cycle (CT).

melt curve A plot of data collected during the melt curve stage. Peaks in the melt curve can
indicate the melting temperature (Tm) of the target or can identify nonspecific PCR
amplification. In the StepOne™ software, you can view the melt curve as normalized
reporter (Rn) vs. temperature or as derivative reporter (−Rn′) vs. temperature. Also
called dissociation curve.

melt curve stage In the thermal profile, a stage with a temperature increment to generate a melt curve.

melting In melt curve experiments, the temperature at which 50% of the DNA is double-
temperature (Tm) stranded and 50% of the DNA is dissociated into single-stranded DNA. The Tm is
displayed in the melt curve.

multicomponent A plot of the complete spectral contribution of each dye for the selected well(s) over
plot the duration of the PCR run.

negative control In the StepOne™ software, the task for targets or SNP assays in wells that contain
(NC) water or buffer instead of sample. No amplification of the target should occur in
negative control wells. Previously called no template control (NTC).

negative control- In presence/absence experiments, wells that contain IPC blocking agent instead of
blocked IPC wells sample in the PCR reaction. No amplification should occur in negative control-
blocked IPC wells because the reaction contains no sample and amplification of the
IPC is blocked. Previously called no amplification control (NAC).

negative control- In presence/absence experiments, wells that contain IPC template and buffer or water
IPC wells instead of sample. Only the IPC template should amplify in negative control-IPC wells
because the reaction contains no sample. Previously called IPC+.

no amplification See negative control-blocked IPC wells.

control (NAC)

Glossary-6 Applied Biosystems StepOne™ and StepOnePlus™ Real-Time PCR Systems Reagent Guide
 Glossary

no template control See negative control (NC).

(NTC)

nonfluorescent Molecules that are attached to the 3′ end of TaqMan® probes. When the probe is intact,
quencher-minor the nonfluorescent quencher (NFQ) prevents the reporter dye from emitting
groove binder fluorescence signal. Because the NFQ does not fluoresce, it produces lower
(NFQ-MGB) background signals, resulting in improved precision in quantitation. The minor groove
binder (MGB) increases the melting temperature (Tm) without increasing probe
length. It also allows the design of shorter probes.

normalized Quantity of target divided by the quantity of endogenous control.

quantity

normalized reporter Fluorescence signal from the reporter dye normalized to the fluorescence signal of the
(Rn) passive reference.

omit well An action that you perform before reanalysis to omit one or more wells from analysis.
Because no algorithms are applied to omitted wells, omitted wells contain no results.

outlier For a set of data, a datapoint that is significantly smaller or larger than the others.

passive reference A dye that produces fluorescence signal. Because the passive reference signal should
be consistent across all wells, it is used to normalize the reporter dye signal to account
for non-PCR related fluorescence fluctuations caused by minor well-to-well
differences in concentrations or volume. Normalization to the passive reference signal
allows for high data precision.

plate layout An illustration of the grid of wells and assigned content in the reaction plate. In
StepOne™ systems, the grid contains 6 rows and 8 columns. In StepOnePlus™
systems, the grid contains 8 rows and 12 columns.
In the StepOne™ software, you can use the plate layout as a selection tool to assign
well contents, to view well assignments, and to view results. The plate layout can be
printed, included in a report, exported, and saved as a slide for a presentation.

point One standard in a standard curve. The standard quantity for each point in the standard
curve is calculated based on the starting quantity and serial factor.

positive control In genotyping experiments, a DNA sample with a known genotype, homozygous or
heterozygous. In the StepOne™ software, the task for the SNP assay in wells that
contain a sample with a known genotype.

post-PCR read Used in genotyping and presence/absence experiments, the part of the instrument run
that occurs after amplification. In genotyping experiments, fluorescence data
collected during the post-PCR read are displayed in the allelic discrimination plot and
used to make allele calls. In presence/absence experiments, fluorescence data
collected during the post-PCR read are displayed in the presence/absence plot and
used to make detection calls. Also called endpoint read.

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Glossary

pre-PCR read Used in genotyping and presence/absence experiments, the part of the instrument run
that occurs before amplification. The pre-PCR read is optional but recommended.
Fluorescence data collected during the pre-PCR read can be used to normalize
fluorescence data collected during the post-PCR read.

primer mix PCR reaction component that contains the forward primer and reverse primer
designed to amplify the target.

primer/probe mix PCR reaction component that contains the primers designed to amplify the target and
a TaqMan® probe designed to detect amplification of the target.

pure dye See custom dye and system dye.

quantitation In quantitation experiments, the method used to determine the quantity of target in the
method samples. In StepOne™ and StepOnePlus™ systems, there are three types of
quantitation methods: standard curve, relative standard curve, and comparative CT
(ΔΔCT).

quantity In quantitation experiments, the amount of target in the samples. Absolute quantity
can refer to copy number, mass, molarity, or viral load. Relative quantity refers to the
fold-difference between normalized quantity of target in the sample and normalized
quantity of target in the reference sample.

quencher A molecule attached to the 3′ end of TaqMan® probes to prevent the reporter from
emitting fluorescence signal while the probe is intact. With TaqMan® reagents, a
nonfluorescent quencher-minor groove binder (NFQ-MGB) can be used as the
quencher. With SYBR® Green reagents, no quencher is used.
IMPORTANT! Applied Biosystems does not recommend the use of TAMRA™ dye as
reporter or quencher with the StepOne™ system. TAMRA dye may be used as a
reporter or quencher with the StepOnePlus™ system.

QuickStart A feature in StepOne™ and StepOnePlus™ systems that allows you to run an
experiment without entering plate setup information. QuickStart requires a colocated
layout with the instrument powered on and an intact instrument-computer connection.

R2 value Regression coefficient calculated from the regression line in the standard curve. The
R2 value indicates the closeness of fit between the standard curve regression line and
the individual CT data points from the standard reactions. A value of 1.00 indicates a
perfect fit between the regression line and the data points.

ramp The rate at which the temperature changes during the instrument run. Except for the
melt curve step, the ramp is defined as a percentage. For the melt curve step, the ramp
is defined as a temperature increment. In the graphical view of the thermal profile, the
ramp is indicated by a diagonal line.

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 Glossary

ramp speed Speed at which the temperature ramp occurs during the instrument run. Available
ramp speeds include fast and standard.
• For optimal results using the fast ramp speed, Applied Biosystems recommends
using TaqMan® Fast reagents in your PCR reactions.
• For optimal results using the standard ramp speed, Applied Biosystems
recommends using standard reagents in your PCR reactions.
IMPORTANT! TaqMan Fast reagents are not supported for genotyping or
presence/absence experiments.

raw data plot A plot of raw fluorescence signal (not normalized) for each optical filter.

reaction mix A solution that contains all components to run the PCR reaction, except for the
template (sample, standard, or control).

reagents The PCR reaction components you are using to amplify the target and to detect
amplification. Types of reagents used on the StepOne™ and StepOnePlus™ systems:
• TaqMan® reagents
• SYBR® Green reagents
• Other reagents

real-time PCR Process of collecting fluorescence data during PCR. Data from the real-time PCR are
used to calculate results for quantitation experiments or to troubleshoot results for
genotyping or presence/absence experiments.

reference sample In relative standard curve and comparative CT (ΔΔCT) experiments, the sample used
as the basis for relative quantitation results. Also called the calibrator.

refSNP ID Identifies the reference SNP (refSNP) cluster ID. Generated by the Single Nucleotide
Polymorphism Database of Nucleotide Sequence Variation (dbSNP) at the National
Center for Biotechnology Information (NCBI). The refSNP ID can be used to search
the Applied Biosystems Store for an Applied Biosystems SNP Genotyping Assay.
Also called an rs number.

regression Values calculated from the regression line in standard curves, including the R2 value,
coefficients slope, and y-intercept. You can use the regression coefficients to evaluate the quality
of results from the standards. See also standard curve.

regression line In standard curve and relative standard curve experiments, the best-fit line from the
standard curve. Regression line formula:
CT = m [log (Qty)] + b
where m is the slope, b is the y-intercept, and Qty is the standard quantity.
See also regression coefficients.

reject well An action that the software performs during analysis to remove one or more wells
from further analysis if a specific flag is applied to the well. Rejected wells contain
results calculated up to the point of rejection.

Applied Biosystems StepOne™ and StepOnePlus™ Real-Time PCR Systems Reagent Guide Glossary-9
Glossary

relative standard Method for determining relative target quantity in samples. With the relative standard
curve method curve method, the StepOne™ software measures amplification of the target and of the
endogenous control in samples, in a reference sample, and in a standard dilution
series. Measurements are normalized using the endogenous control. Data from the
standard dilution series are used to generate the standard curve. Using the standard
curve, the software interpolates target quantity in the samples and in the reference
sample. The software determines the relative quantity of target in each sample by
comparing target quantity in each sample to target quantity in the reference sample.

Remote Monitor A feature in the StepOne™ software that allows you to monitor a StepOne™ or
StepOnePlus™ instrument over the network. With the Remote Monitor, you can
monitor the instrument status, send an experiment to the instrument, monitor
amplification plots and temperature plots in real time, and download the results to
your computer. You cannot operate the StepOne or StepOnePlus instrument using the
Remote Monitor.

replicate group A set of identical reactions in an experiment.

replicates Total number of identical reactions containing identical components and identical
volumes.

reporter Fluorescent dye used to detect amplification. If you are using TaqMan® reagents, the
reporter dye is attached to the 5′ end. If you are using SYBR® Green reagents, the
reporter dye is SYBR® Green dye.

reverse primer An oligonucleotide that flanks the 3′ end of the amplicon. The reverse primer and the
forward primer are used together in PCR reactions to amplify the target.

reverse An enzyme that converts RNA to cDNA. Reverse transcriptase is added to the PCR
transcriptase reaction to perform 1-step RT-PCR.

Rn See normalized reporter (Rn).

ROX™ dye A dye supplied by Applied Biosystems and precalibrated on the StepOne™ and
StepOnePlus™ systems. ROX dye is used as the passive reference.

rs number See refSNP ID.

run method Definition of the reaction volume and the thermal profile for the StepOne™ or
StepOnePlus™ instrument run.

sample The template that you are testing.

Sample DNA (10✕) In the StepOne™ software, a reaction component displayed on the Reaction Mix
Calculations tab of the Reaction Setup screen. The software assumes the sample DNA
is added to the reaction mix at a 10✕ concentration. For example, if the reaction
volume is 20 μL, the calculated volume of sample for 1 reaction is 2 μL.

Sample Library In the StepOne™ software, a collection of samples. The Sample Library contains the
sample name and the sample color.

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 Glossary

Sample or In the StepOne™ software, a reaction component displayed on the Reaction Mix
Standard (10✕) Calculations tab of the Reaction Setup screen. The software assumes the sample or
standard is added to the reaction mix at a 10✕ concentration. For example, if the
reaction volume is 20 μL, the calculated volume of sample or standard for 1 reaction
is 2 μL.

sample/SNP assay In genotyping experiments, the combination of which sample to test and which SNP
reaction assay to perform in one PCR reaction. Each PCR reaction can contain only one sample
and one SNP assay.

sample/target In quantitation experiments, the combination of which sample to test and which target
reaction to detect and quantify in one PCR reaction. In the Design Wizard, you can detect and
quantify only one target in one PCR reaction. Use Advanced Setup to detect and
quantify more than one target in one PCR reaction.

serial factor In the StepOne™ software, a numerical value that defines the sequence of quantities
in the standard curve. The serial factor and the starting quantity are used to calculate
the standard quantity for each point in the standard curve. For example, if the
standard curve is defined with a serial factor of 1:10 or 10✕, the difference between
any 2 adjacent points in the curve is 10-fold.

series See standard dilution series.

slope Regression coefficient calculated from the regression line in the standard curve. The
slope indicates the PCR amplification efficiency for the assay. A slope of −3.32
indicates 100% amplification efficiency. See also amplification efficiency (EFF%)
and regression line.

SNP Abbreviation for single nucleotide polymorphism. The SNP can consist of a base
difference or an insertion or deletion of one base.

SNP assay Used in genotyping experiments, a PCR reaction that contains primers to amplify the
SNP and two probes to detect different alleles.

SNP Assay Library In the StepOne™ software, a collection of SNP assays to add to genotyping
experiments. The SNP assays in the library contain the SNP assay name, SNP assay
color, and for each allele, the allele name or base(s), reporter, quencher, and allele
colors. The SNP assays in the library may also contain the assay ID and comments
about the SNP assay.

spatial calibration Type of StepOne™ and StepOnePlus™ system calibration in which the system maps
the positions of the wells in the sample block(s). Spatial calibration data are used so
that the software can associate increases in fluorescence during a run with specific
wells in the reaction plate.

stage In the thermal profile, a group of one or more steps. There are three types of stages:
holding stage (including pre-PCR read and post-PCR read), cycling stage (also called
amplification stage), and melt curve stage.

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Glossary

standalone layout A system layout in which the StepOne™ or StepOnePlus™ instrument is not connected
to a computer by the yellow cable. In this layout, you control the instrument only with
the instrument touchscreen, and you use a USB drive or network connection to
transfer data between the instrument and computer.

standard Sample that contains known standard quantities. Standard reactions are used in
quantitation experiments to generate standard curves. See also standard curve and
standard dilution series.

standard curve In standard curve and relative standard curve experiments:

• The best-fit line in a plot of the CT values from the standard reactions plotted
against standard quantities. See also regression line.
• A set of standards containing a range of known quantities. Results from the
standard curve reactions are used to generate the standard curve. The standard
curve is defined by the number of points in the dilution series, the number of
standard replicates, the starting quantity, and the serial factor. See also standard
dilution series.

standard curve Method for determining absolute target quantity in samples. With the standard curve
method method, the StepOne™ software measures amplification of the target in samples and
in a standard dilution series. Data from the standard dilution series are used to
generate the standard curve. Using the standard curve, the software interpolates the
absolute quantity of target in the samples. See also standard and standard curve.

standard dilution In standard curve and relative standard curve experiments, a set of standards
series containing a range of known quantities. The standard dilution series is prepared by
serially diluting standards. For example, the standard stock is used to prepare the first
dilution point, the first dilution point is used to prepare the second dilution point, and
so on. In the StepOne™ software, the volumes needed to prepare a standard dilution
series are calculated by the number of dilution points, the number of standard
replicates, the starting quantity, the serial factor, and the standard concentration in the
stock. See also standard curve.

standard quantity A known quantity in the PCR reaction.

• In standard curve experiments, the quantity of target in the standard. In the
StepOne™ software, the units for standard quantity can be for mass, copy
number, viral load, or other units for measuring the quantity of target.
• In relative standard curve experiments, a known quantity in the standard.
Standard quantity can refer to the quantity of cDNA or the quantity of standard
stock in the PCR reaction. The units are not relevant for relative standard curve
experiments because they cancel out in the calculations.

starting quantity When defining a standard curve in the StepOne™ software, corresponds to the highest
or lowest quantity.

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 Glossary

step A component of the thermal profile. For each step in the thermal profile, you can set
the ramp rate (ramp increment for melt curve steps), hold temperature, hold time
(duration), and you can turn data collection on or off for the ramp or the hold parts of
the step. For cycling stages, a step is also defined by the AutoDelta status. With
StepOnePlus™ systems, which contain the VeriFlex™ blocks, each step contains 6
temperatures (1 for each VeriFlex block).

SYBR® Green PCR reaction components that consist of two primers designed to amplify the target
reagents and SYBR® Green dye to detect double-stranded DNA.

system dye Dye supplied by Applied Biosystems and precalibrated on the StepOne™ or
StepOnePlus™ system. Before you use system dyes in your experiments, make sure
the system dye calibration is current in the Instrument Maintenance Manager.
System dyes on the StepOne system:
• FAM™ dye
• JOE™ dye
• ROX™ dye
• SYBR® Green dye
• VIC® dye
System dyes on the StepOnePlus system:
• FAM™ dye
• JOE™ dye
• NED™ dye
• ROX™ dye
• SYBR® Green dye
• TAMRA™ dye
• VIC® dye
IMPORTANT! Applied Biosystems does not recommend the use of TAMRA™ dye as
reporter or quencher with the StepOne™ system. TAMRA dye may be used as a
reporter or quencher with the StepOnePlus™ system.

TaqMan® reagents PCR reaction components that consist of primers designed to amplify the target and a
TaqMan® probe designed to detect amplification of the target.

target The nucleic acid sequence that you want to amplify and detect.

target color In the StepOne™ software, a color assigned to a target to identify the target in the plate
layout and analysis plots.

Target Library In the StepOne™ software, a collection of targets to add to experiments. The targets in
the library contain the target name, reporter, quencher, and target color. The target in
the library may also contain comments about the target.

Applied Biosystems StepOne™ and StepOnePlus™ Real-Time PCR Systems Reagent Guide Glossary-13
Glossary

task In the StepOne™ software, the type of reaction performed in the well for the target or
SNP assay. Available tasks:
• Unknown
• Negative Control
• Standard (standard curve and relative standard curve experiments)
• Positive control (genotyping experiments)
• IPC (presence/absence experiments)
• Blocked IPC (presence/absence experiments)

temperature plot In the StepOne™ software, a display of temperatures for the sample, instrument cover,
and instrument block during the StepOne™ or StepOnePlus™ instrument run.

template In the Design Wizard of the StepOne™ software (and in QuickStart for quantitation
experiments), the type of nucleic acid to add to the PCR reaction. The recommended
template varies according to experiment type:
• Quantitation experiments (standard curve, relative standard curve, and
comparative CT) – cDNA (complementary cDNA), RNA, or gDNA (genomic
DNA)
For quantitation experiments, the template type selection affects the run method,
reaction setup, and materials list.
• Genotyping experiments – Wet DNA (gDNA or cDNA) or dry DNA (gDNA or
cDNA)
For genotyping experiments, the template type selection affects the reaction
setup.
• Presence/absence experiments - DNA
For presence/absence experiments, Applied Biosystems recommends adding
DNA templates to the PCR reactions.

thermal profile Part of the run method that specifies the temperature, time, ramp, and data collection
points for all steps and stages of the StepOne™ or StepOnePlus™ instrument run.

threshold 1. In amplification plots, the level of fluorescence above the baseline and within
the exponential growth region The threshold can be determined automatically
(see automatic CT) or can be set manually (see manual CT).

2. In presence/absence experiments, the level of fluorescence above which the

StepOne™ software assigns a presence call.

threshold cycle (CT) The PCR cycle number at which the fluorescence meets the threshold in the
amplification plot.

Tm See melting temperature (Tm).

touchscreen Instrument display that you touch to control the StepOne™ or StepOnePlus™
instrument.

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 Glossary

unknown In the StepOne™ software, the task for the target or SNP assay in wells that contain
the sample you are testing:
• In quantitation experiments, the task for the target in wells that contain a sample
with unknown target quantities.
• In genotyping experiments, the task for the SNP assay in wells that contain a
sample with an unknown genotype.
• In presence/absence experiments, the task for the target in wells that contain a
sample in which the presence of the target is not known.

unknown-IPC wells In presence/absence experiments, wells that contain a sample and internal positive
control (IPC).

VeriFlex™ The StepOnePlus™ instrument contains six independently thermally regulated

Technology VeriFlex™ blocks, creating up to six different zones for the 96 sample wells. After you
enable the VeriFlex blocks in the StepOne™ software, you can set a different
temperature for one or more of the VeriFlex blocks.

y-intercept In the standard curve, the value of y where the regression line crosses the y-axis. The
y-intercept indicates the expected threshold cycle (CT) for a sample with quantity
equal to 1.

zone One of up to six sample temperatures among the 96 wells formed by independently
thermally regulated VeriFlex™ blocks during the StepOnePlus™ instrument run. You
can set a different temperature for one or more of the VeriFlex blocks, or you can set
the same temperature for each of the VeriFlex blocks.

Note: For melt curve steps, you need to set the same temperature for each of the
VeriFlex blocks.

zone boundary The edge of a zone for samples formed by the six independently thermally regulated
VeriFlex™ blocks. In the StepOne™ software, the zone boundaries are displayed in the
plate layout as thick red lines.

Applied Biosystems StepOne™ and StepOnePlus™ Real-Time PCR Systems Reagent Guide Glossary-15
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Glossary-16 Applied Biosystems StepOne™ and StepOnePlus™ Real-Time PCR Systems Reagent Guide
Index

Numerics Also see materials required 1-4

supported 1-4
1-step RT-PCR
contamination, minimizing DNA 2-7
about 3-8
primers used 3-9 Custom assay type 1-9, 1-10
RNA quantitation 3-25, D-4, D-9 presence/absence experiments 5-16
quantitation experiments 3-21
2-step RT-PCR
about 3-8 Custom TaqMan Gene Expression Assays 3-18, 5-12
primers used 3-9 Custom TaqMan SNP Genotyping Assays 4-14
RNA quantitation 3-26, D-4, D-9
D
A data
Advanced Setup workflow 1-9, 1-10 about data collection 1-2
amplicon sites Design Wizard workflow 1-9, 1-10
3’ primer end 3-23 designing experiments
5’ probe end 3-22 genotyping 4-14, 4-16
G/C content 3-22 presence/absence 5-16
melting temperature 3-22 quantitation 3-20, 3-21
screening 3-21 DNA/cDNA quantitation, thermal cycling
selection 3-21 conditions 3-28
amplicons, selecting small 3-22 dye-binding, methods of 2-5
Assay Design Guidelines
about C-1
genotyping experiments C-2 E
optimize primer concentrations 3-30 endogenous control
optimize probe concentration 3-33 component of experiment 3-6
presence/absence experiments 5-16 endpoint experiments
primer and probe 3-21, 3-23 genotyping 4-4
quantitation experiments 3-21, C-1 presence/absence 5-5
selecting reagents 3-24
thermal cycling conditions 3-27
assay types
G
Custom assays 1-9, 1-10 G/C content, and amplicon sites 3-22
genotyping experiments 4-6 genotyping experiments
Inventoried/Made to Order Assays 1-9 assay types 4-6
Pre-Designed/Validated assays 1-10 components 4-4
presence/absence experiments 5-6 conclusions for Assay Design Guidelines C-2
quantitation experiments 3-12 described 4-4
selecting 1-9 designing 4-14, 4-16
how they work 4-4
C instruments 4-4
mismatches 4-5
carryover, UNG to minimize 2-7 selecting master mix 4-13, 4-16
comparative CT experiments TaqMan reagents 2-2
about 3-6
Also see quantitation experiments 3-6
components 3-6
H
consumables hairpin loops, and primer choice 3-9

Applied Biosystems StepOne™ and StepOnePlus™ Real-Time PCR Systems Reagent Guide Index-1
Index

I primer matrix
defining limits B-1
Inventoried assay type 1-9 example of limiting B-2
IPC 5-4, 5-5 how used 3-30
primers
M 1-step RT-PCR 3-9
2-step RT-PCR 3-9
Made to Order assay type 1-9
default concentrations 3-30
master mix hairpin loops 3-9
selecting for genotyping experiments 4-13, 4-16 summary of design guidelines 3-23
selecting for presence/absence experiments 5-15 probes
selecting for quantitation experiments 3-19
available TaqMan MGB probes 2-4
melting temperature, and amplicon sites 3-22 optimizing probe concentration 3-33
mismatch, in genotyping experiments 4-5 summary of design guidelines 3-23
multiplex PCR
described 3-10 Q
primer limiting 3-10
rRNA primers B-1 quantitation experiments
singleplex comparison 3-10 Assay Design Guidelines for Custom assay type 3-21
MultiScribe reverse transcriptase, defined 3-26 assay types 3-12
comparative CT 3-6
comparing 3-7
N conclusions for Assay Design Guidelines C-1
negative control, component of experiment 3-5, 3-6, 5-4 designing 3-20, 3-21
negative controls 4-4 explained 3-4
real-time PCR 3-4
nonspecific product, contamination with SYBR Green relative standard curve 3-5
dye 2-7 selecting a quantitation method 3-5
selecting master mix 3-19
O selecting reagent type 3-12
selecting reagents for Custom assay type 3-24, D-3,
optimization 3-33 D-9
other fluorescent-based reagents 1-8 standard curve 3-5
SYBR Green reagents 2-4, 3-31
P TaqMan Reagents 2-2
PCR, general practices 2-8
positive controls 4-4 R
Pre-Designed/Validated assay type 1-10 reagents
presence/absence experiments considerations 2-6
Assay Design Guidelines for Custom assay type 5-16 other fluorescent-based 1-8
assay types 5-6 selecting 1-8, 2-6
components 5-4 selecting for Custom assay type 3-24, D-3, D-9
defined 5-4 SYBR Green reagents 1-8
designing 5-16 TaqMan reagents 1-8, 2-2
how they work 5-5 real-time PCR
incorporating an IPC 5-5 quantitation experiments 3-4
performing without an IPC 5-4 TaqMan detection process 2-2
selecting master mix 5-15 reference sample
selecting reagents for Custom assay type 3-24, D-3, component of experiment 3-5, 3-6
D-9 relative standard curve experiments
TaqMan reagents 2-2 about 3-5
Primer Express Software Also see quantitation experiments 3-5
presence/absence experiments 5-16 components 3-5
quantitation experiments 3-21 replicate, component of experiment 3-5, 3-6
small amplicons 3-22 replicates 4-4, 5-4
SNP assays 1-10
RNA quantitation
primer limiting, multiplex assays 3-10

Index-2 Applied Biosystems StepOne™ and StepOnePlus™ Real-Time PCR Systems Reagent Guide
 Index

1-step RT-PCR 3-25, D-4, D-9 U

2-step RT-PCR 3-26, D-4, D-9
thermal cycling conditions 3-28, 3-29 UNG, and minimizing carryover 2-7
RT-PCR Universal Master Mix reagents
1-step 3-8 polymerase benefit 3-26
2-step 3-8
method comparison 3-7, 3-9

S
sample 4-4, 5-4
samples
component of experiment 3-5, 3-6
small amplicons, selecting 3-22
standard curve experiments
about 3-5
Also see quantitation experiments 3-5
components 3-5
standard dilution series
component of experiment 3-5, 3-6
standards
component of experiment 3-5, 3-6
StepOne system
assay types 1-9
consumables 1-4
data collection 1-2
experiment types 1-7
filters 1-3
reagent types 1-8
SYBR Green reagents 1-3
considerations for selecting 2-6
development 2-5
how they work 2-5
optimizing quantitation experiments 3-31

T
TaqMan Drug Metabolism Genotyping Assays 4-11
TaqMan Endogenous Control Assays 3-17
TaqMan Exogenous Internal Positive Control
Reagents 5-13
TaqMan Gene Expression Assays 3-16, 5-10
TaqMan MGB probes 2-3
usage 2-4
TaqMan PDARs for AD 4-12
TaqMan reagents 1-3
considerations for selecting 2-6
development 2-2
experiment types 2-2
how they work 2-2
TaqMan SNP Genotyping Assays 4-10
thermal cycling conditions
1-step RT-PCR 3-28
2-step RT-PCR 3-29
DNA/cDNA quantitation 3-28
RNA quantitation 3-28

Applied Biosystems StepOne™ and StepOnePlus™ Real-Time PCR Systems Reagent Guide Index-3
Index

Index-4 Applied Biosystems StepOne™ and StepOnePlus™ Real-Time PCR Systems Reagent Guide
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