Amplification: MyiQ and iQ5 Real-Time PCR Systems

MyiQ™ and iQ™5

Real-Time PCR Detection Systems
Genomic Research Solutions
From Bio-Rad
Bio-Rad is well known for making advanced genomic technologies
accessible to every researcher. In addition to producing outstanding
instrumentation for real-time PCR detection, Bio-Rad offers a variety of
solutions for simplifying everything from assay design to gene expression
analysis. This complete collection of tools provides powerful building
blocks for your genomic discoveries.

Bio-Rad Offers Real-Time Instruments Designed

for the Way You’d Rather Work
n Modular instruments with high-resolution optics for maximum flexibility and sensitivity
n Gradient functionality for rapid assay optimization
n Outstanding thermal performance delivered by the iCycler® thermal cycler
n PCR supermixes that provide optimum performance in real-time PCR assays
n  recisely manufactured plates, tubes, and sealers tested for optimal fit and
P
performance in Bio-Rad real-time PCR instruments
Products for Your
Genomics Workflow

Beacon Designer software facilitates the design and

Assay
selection of primers and probes for a variety of real-time
Design
PCR applications.

Sample Aurum™ RNA isolation kits yield fast results with the
Preparation highest level of sample purity.

Sample Quality The Experion™ automated electrophoresis system quickly

Assessment and accurately measures RNA quality.

Real-Time A broad variety of PCR reagents and instruments

Amplification provide the flexibility and reliability you need to accelerate
and Analysis discovery.
 Smart Modular Designs Emission filter wheel
CCD

The MyiQ and iQ5 real-time PCR detection systems are

96-well modular upgrades to the iCycler thermal cycler,
Fold mirror
which delivers excellent thermal performance and
thermal gradient capability. Sensitive charge-coupled
device (CCD)-based optics enable accurate quantitation
over a dynamic range of 9 orders of magnitude.

MyiQ System
 he MyiQ system offers an affordable option for the
T
detection of common green fluorescent dyes, such as
FAM and SYBR Green I.
Excitation
iQ5 System filter wheel
Lens
The iQ5 system offers five-target analysis capabilities for
multiplex PCR with a variety of detection chemistries.
96-well plate
The combined performance of the iCycler thermal cycler,
the CCD-based optics, and Bio-Rad supermixes generates
exceptionally uniform results across a 96-well block. With Optical system. Excitation of all 96 wells is achieved by a
reaction volumes as low as 10 µl, the iQ5 and MyiQ systems combination of narrow-bandpass filters and a tungsten-halogen
lamp. Filtered light from the lamp is reflected off mirrors, passes
produce uniform data with standard deviations of <0.15 through a condensing lens, and is focused into the center of each
cycles (see data below). well. Fluorescent light emitted from the wells reflects off the main
fold mirror, passes through an emission filter, and is detected by a
12-bit CCD.

Volume 10 µl Volume 25 µl
103 Avg. CT 19.84 Avg. CT 20.97
SD 0.11 SD 0.11
PCR baseline-subtracted curve fit RFU

103

102
102

0 5 10 15 20 25 30 35 40 0 5 10 15 20 25 30 35 40
Cycle Cycle

Excellent uniformity. IL1-b plasmid template was diluted to 105 copies/reaction and amplified in the presence of a FAM-labeled detection probe
with iQ™ supermix using an iQ5 real-time PCR detection system. Left panel, replicates of 10 µl reactions. Right panel, replicates of 25 µl reactions.
Inset: CT, threshold cycle; SD, standard deviation.
 Outstanding Gradient Performance
Optimize Reactions in a
Single Experiment
Bio-Rad real-time systems offer a thermal
gradient feature, which allows you to optimize
assays in a single experiment by using a range
of temperatures simultaneously. At any step in a
protocol, a temperature gradient of up to 25°C
may be programmed across the reaction block.
n Gradient control provides exceptional
uniformity and reproducibility within each zone
n  asy programming with onscreen presentation
E
of gradient temperatures
n Precision ramping
n Available for both conventional and
quantitative PCR instrumentation
n  ach temperature within the thermal gradient
E
Thermal gradient. Optimizing incubation temperatures for real-time PCR assays is critical,
is listed in validation reports particularly for multiplex assays, but it is not always easy to do. After the melting temperature (Tm)
of a primer is calculated, the annealing temperature must be determined empirically for optimal
results. This often involves repeating a reaction at many different temperatures. Similar time-
consuming tests may be required to optimize the denaturation temperature. The thermal gradient
feature facilitates identification of the most favorable temperatures for optimal assay performance.

2,500
PCR baseline-subtracted curve fit RFU

2,000

1,500

1,000

500
37.64

0
0 5 10 15 20 25 30 35 40 45 50
Cycle

Thermal gradient experiment for optimizing annealing temperature. A 10-fold dilution series (108 –102 copies) of
plasmid containing GAPDH template was amplified using a thermal gradient in the presence of SYBR Green I dye. The
amplification reaction was monitored and analyzed with an iQ5 real-time PCR detection system. Eight identical reactions
were prepared for each template concentration, one for each of the eight PCR annealing temperatures, ranging from
55 to 70°C. An optimal primer annealing temperature of 58°C (shown in the green traces), which resulted in the earliest
CT value, was identified in this gradient assay. The blue traces show the results for 64.5°C as a comparison.
Efficient, Accurate, Easy-to-Use Software
Making the most of a powerful real-time PCR system
requires a flexible and easy-to-use software package.
The iQ5 optical system software meets this need with
quick-setup tools, a full suite of analysis tools, and a
variety of presentation options. In addition, iQ5 optical
system software, Security Edition, provides the tools
required to meet regulatory requirements.
Standard features of iQ5 software include:
n  dvanced gene expression analysis options, including
A
comparison to multiple reference genes, correction for
reaction efficiency, and more
n Ability to export any data collected and analyzed by iQ5
software directly to a Microsoft Excel spreadsheet
n Gene Study module for multifile gene expression
analysis to directly compare over 5,000 CT values from
iQ5 and MyiQ data
Multifile gene expression analysis. Compare CT values from different
n  ser Profile management tool for creating unique user
U data files with the Gene Study in iQ5 software. Over 5,000 CT values
can be analyzed in a single Gene Study file.
log-in names, with optional password protection, for file
storage and data analysis preferences
n  uilt-in analysis modules for absolute quantitation,
B
melt-curve analysis, end-point analysis, and allelic
discrimination (for multiplex data)

Security for Those Who Need It

The Security Edition of iQ5 optical system software can
integrate the power of an iQ5 real-time PCR detection
system with your laboratory’s good laboratory practice
(GLP) standards for data collection and data analysis. This
software is designed for routine use by scientists who
operate in regulated environments that require electronic
record-keeping procedures.
The Security Edition offers several major functions and
features, including:
n  mbedded tools for compliance with US FDA 21 CFR
E
Part 11 regulations, including electronic signature and
added traceability features
n  nhanced reporting tools for customizing the secure
E
presentation of assay results

Electronic record-keeping tools. The iQ5 Security Edition software

enables electronic signatures to be applied to calibration and data files
generated by iQ5 or MyiQ systems.
Optimized Reagents for Peak Performance
Bio-Rad reagents accentuate the performance of A. FAM (b-actin) B. HEX (GAPDH)

PCR baseline-subtracted curve fit RFU

PCR baseline-subtracted curve fit RFU
your real-time system. All reagents demonstrate high
performance with minimal optimization over a wide 103 103
dynamic range for input RNA, cDNA, genomic DNA,
and plasmid DNA. 127.52
103.47
102 102

Detect Multiple Targets Without Optimization

iQ multiplex powermix is a robust mix that greatly 0 10 20 30 40 50 0 10 20 30 40 50
Cycle Cycle
simplifies the challenge of identifying reaction conditions
C. Texas Red (IL-1b) D. Cy5 (a-tubulin)
that will amplify targets with equal efficiency in singleplex

PCR baseline-subtracted curve fit RFU

PCR baseline-subtracted curve fit RFU

and multiplex real-time PCR reactions. This mix makes 103
multiplex real-time PCR easier by removing the need to 103
optimize buffer, enzyme, or primer concentrations.
144.82
102
Convenient One-Step RT-qPCR for 49.56
102

Any Detection Chemistry

iScript™ one-step RT-PCR kits are optimized to deliver 0 10 20 30 40 50 0 10 20 30 40 50
Cycle Cycle
maximum RT-PCR efficiency, sensitivity, and specificity.
E. TAMRA (cyclophilin) F. Standard curves
These kits contain a proprietary reaction buffer that has
PCR baseline-subtracted curve fit RFU

45
• Standard
been specifically formulated to optimize activity of both 40
6 Unknown
35
iScript reverse transcriptase and iTaq™ DNA polymerase, 103
30
while minimizing the potential for primer-dimer formation 308.78
25

CT
and other nonspecific PCR artifacts. With these kits, 20
15
clean detection of low-copy targets is easy to achieve. 102
10
5
Protect Precious Samples With 0 10 20 30 40 50 0 2 4 6 8 10
Cycle Starting quantity, copy number
High-Quality Plastic Consumables
Linearity of five-target multiplex detection with the iQ5 real-time PCR detection
Bio-Rad offers high-quality consumables for a wide
system. A–E, fluorescence data from a series of 10-fold dilutions of plasmid DNA
variety of applications, backed by technical support (108–102 copies per 25 µl reaction) amplified using five reporter dyes to monitor five
professionals. targets, as indicated above panels. F, standard curves generated from data in A–E:
FAM, R2 = 0.998, efficiency = 98.0%; HEX, R2 = 0.999, efficiency = 104.4%; Texas
Each box of Bio-Rad tubes, plates, and caps is Red, R2 = 0.999, efficiency = 102.5%; Cy5, R2 = 0.999, efficiency = 98.9%; TAMRA,
R2 = 0.999, efficiency = 100.8%.
process-sampled and tested to be negative for DNase,
RNase, and DNA. All reaction vessels and sealing
systems have been designed to provide the best
PCR baseline-subtracted curve fit RFU

possible fit and performance with a corresponding

Bio-Rad thermal cycler. For real-time PCR applications,
1,000
Bio-Rad offers precisely manufactured tubes, plates,
sealers, and accessories that have been tested for
optimal performance in iQ5 and MyiQ systems.

100

0 5 10 15 20 25 30 35 40 45 50
Cycle

An accurate one-cycle spacing between CT values is precisely maintained in a

series of 2-fold dilutions. Human genomic DNA (120–15 ng) was amplified with
iQ supermix using primers and a probe specific to the IL-1b gene. Eight replicates
at each template concentration were amplified along with no-template controls
on the MyiQ real-time system. Standard curve had r = –0.999, slope = –3.378,
iQ™ SYBR® Green supermix. efficiency = 97.7%.
 System Specifications Catalog # Description
TBS-0201 0.2 ml 8-Tube Strips Without Caps, natural, 125
Temperature TCS-0803 Optical Flat 8-Cap Strips, for 0.2 ml tubes and plates,
Range 4–100°C ultraclear, 120
Accuracy ±0.3°C of programmed target MSB-1001 Microseal 'B' Adhesive Seals, 100
Uniformity ±0.4°C well-to-well 170-8848 iQ Multiplex Powermix, 50 x 50 µl reactions, 2x mix
contains dNTPs (including dUTP), 11 mM MgCl2, iTaq
Gradient Mode
DNA polymerase, stabilizers
Gradient accuracy ±0.4°C
170-8860 iQ Supermix, 100 x 50 µl reactions, 2x mix contains
Row uniformity ±0.4°C
100 mM KCl, 40 mM Tris-HCl, pH 8.4, 0.4 mM each
Gradient range 40–100°C
dNTP (dATP, dCTP, dGTP, dTTP), 50 U/ml iTaq DNA
Temperature differential 1–25°C
polymerase, 6 mM MgCl2, stabilizers
range
170-8875 iTaq DNA Polymerase, 5 U/µl, includes 5,000 U
Optical System polymerase, 25 ml of 10x PCR buffer, 25 ml of 50 mM
Light source Tungsten-halogen lamp MgCl2 solution
170-8894 iScript One-Step RT-PCR Kit for Probes, 50 x 50 µl
Fluorescence excitation range
reactions, includes iScript reverse transcriptase for
MyiQ 475–495 nm
one-step RT-PCR, 2x probes RT-PCR reaction mix,
iQ5 475–645 nm
nuclease-free water
Fluorescence detection range 732-6800 Aurum Total RNA 96 Kit, 2 x 96-well preps, includes
MyiQ 515–545 nm 2 grow blocks, 2 growth membranes, sealing tape,
iQ5 515–700 nm 2 RNA binding plates, 2 collection microplates, 2 vials
Sensitivity  ne copy of IL-1b in human
O lyophilized DNase I, RNase-free reagents, protocol
genomic DNA overview, instructions
700-7000 Experion System, 100–120/220–240 V, for protein
Dynamic range 9 orders of magnitude
analysis, includes electrophoresis station, priming
Compatible dyes station, software, USB2 cable, instructions (analysis kits
MyiQ FAM, SYBR Green I sold separately)
iQ5 FAM, SYBR Green I, HEX, TAMRA,
Texas Red, Cy5
Beacon Designer is a trademark of PREMIER Biosoft International. Cy is a
Descriptive trademark of GE Healthcare. Excel and Microsoft are trademarks of Microsoft
Sample capacity 96 wells Corp. SYBR and Texas Red are trademarks of Molecular Probes, Inc.
Sample volume 15–100 µl Bio-Rad Laboratories, Inc. is licensed by Molecular Probes, Inc. to sell
Dimensions (W x D x H) 29.2 x 58.4 x 38.7 cm (11.5 x 23 x 15.2") reagents containing SYBR Green I for use in real-time PCR, for research
Weight 17.6 kg (39 lb) purposes only.
Molecular beacons and their use are licensed under patents owned by
The Public Health Research Institute of the City of New York, Inc.
Ordering Information
Notice regarding Bio-Rad thermal cyclers and real-time systems.
Catalog # Description Purchase of this instrument conveys a limited non-transferable immunity from
170-9770 MyiQ Single-Color Real-Time PCR Detection suit for the purchaser’s own internal research and development and for use in
System, includes iCycler base, optics module, applied fields other than Human In Vitro Diagnostics under one or more of U.S.
software CD-ROM, 96-well optical reaction module, Patents Nos. 5,656,493, 5,333,675, 5,475,610 (claims 1, 44, 158, 160–163,
optical-quality 96-well PCR plates, Microseal® 'B' seals, and 167 only), and 6,703,236 (claims 1–7 only), or corresponding claims
communication cables, power cords, instructions in their non-U.S. counterparts, owned by Applera Corporation. No right is
170-9780 iQ5 Multicolor Real-Time PCR Detection System, conveyed expressly, by implication or by estoppel under any other patent claim,
includes iCycler base, optics module, software such as claims to apparatus, reagents, kits, or methods such as 5' nuclease
CD-ROM, 5 installed filter sets, 96-well reaction module, methods. Further information on purchasing licenses may be obtained by
calibration solutions, optical-quality 96-well PCR plates, contacting the Director of Licensing, Applied Biosystems, 850 Lincoln Centre
Microseal 'B' seals, communication cables, power cord, Drive, Foster City, California 94404, USA.
quick reference cards, instructions Bio-Rad’s real-time thermal cyclers are licensed real-time thermal cyclers
170-8734 Beacon Designer Probe/Primer Design Software, under Applera’s United States Patent No. 6,814,934 B1 for use in research
includes CD-ROM, quick guide, instructions and for all other fields except the fields of human diagnostics and veterinary
170-9753SE01 iQ5 Optical System Software, Security Edition, diagnostics.
single-seat license LabChip and the LabChip logo are trademarks of Caliper Life Sciences,
170-9799 Real-Time PCR Applications Guide Inc. Bio-Rad Laboratories, Inc. is licensed by Caliper Life Sciences, Inc.
223-9441 iQ 96-Well PCR Plates, 25 to sell products using the LabChip technology for research use only.

Bio-Rad
Laboratories, Inc.

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