One-Color

Microarray-Based Gene
Expression Analysis
Low Input Quick Amp
Labeling

Protocol
For use with Agilent Gene Expression oligo microarrays

Version 6.9.1, December 2015

Before you begin, view hands-on
videos of SurePrint procedures at Microarrays manufactured with Agilent SurePrint
[Link]
Technology

For Research Use Only. Not for use in diagnostic procedures.

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One-Color Microarray-Based Gene Expression Analysis (Low Input Quick Amp Labeling) Protocol
In this Guide...
This document describes the Agilent recommended procedures
to prepare and label complex biological targets and
hybridization, washing, scanning, and feature extraction of
Agilent 60-mer oligonucleotide microarrays for
microarray-based one-color gene expression analysis.

1 Before You Begin

This chapter contains information (such as procedural notes,
safety information, required reagents and equipment) that you
should read and understand before you start an experiment.

2 Procedures
This chapter describes the steps to prepare samples, hybridize,
wash and scan gene expression microarrays, and to extract data
using the Agilent Feature Extraction Software.

3 Supplemental Procedures
This chapter contains instructions for quality assessment of
template RNA and labeled cRNA, and steps to prevent
ozone-related problems.

4 Reference
This chapter contains reference information related to the
protocol.

One-Color Microarray-Based Gene Expression Analysis (Low Input Quick Amp Labeling) Protocol 3
What’s new in 6.9
• Corrected fragmentation mix table.
• Updated QC spec for specific labeling activity.
• Updated product labeling statement.

What’s new in 6.8

• Updated list of supported microarray and resorted list by
species.
• Expanded instructions to prepare hybridization assembly.

What’s new in 6.7

• Expanded solvent wash details to prepare for microarray
wash.
• Added list of supported microarrays.
• Added note to calibrate hybridization oven on a regular basis
for accuracy of the collected data.
• Corrected the T7 reagent that is used in labeling reaction
preparation step.
• Updated loading instructions for hybridization oven.
• Added reference to compatibility matrix for non-Agilent
scanners.

What’s new in 6.6

• Support for Agilent SureScan microarray scanner.

4 One-Color Microarray-Based Gene Expression Analysis (Low Input Quick Amp Labeling) Protocol
Content

1 Before You Begin 7

Procedural Notes 8
Safety Notes 9
Agilent Oligo Microarrays 10
Required Equipment 13
Required Reagents 15
Optional Equipment/Reagents 16
Required Hardware and Software 16
Optional Software 17

2 Procedures 19
Sample Preparation 21
Step 1. Prepare Spike Mix 23
Step 2. Prepare labeling reaction 26
Step 3. Purify the labeled/amplified RNA 30
Step 4. Quantify the cRNA 32
Hybridization 34
Step 1. Prepare the 10× Blocking Agent 34
Step 2. Prepare hybridization samples 35
Step 3. Prepare the hybridization assembly 37
Step 4. Hybridize 39
Microarray Wash 40
Step 1. Add Triton X-102 to Gene Expression wash buffers 40
Step 2. Prewarm Gene Expression Wash Buffer 2 41
Step 3. Prepare the equipment 41
Step 4. Wash the microarray slides 43
Scanning and Feature Extraction 47
Step 1. Scan the slides 47
Step 2. Extract data using Agilent Feature Extraction Software 50

One-Color Microarray-Based Gene Expression Analysis (Low Input Quick Amp Labeling) Protocol 5
Contents

3 Supplemental Procedures 57
Absolutely RNA Nanoprep Purification 58
Step 1. Prepare the reagents 58
Step 2. Purify the labeled/amplified RNA 59
Thermocycler Protocol 61
Step 1. Program the thermocycler 61
Step 2. Synthesize cDNA from Total RNA 62
Step 3. Synthesize Fluorescent cRNA Synthesis in vitro 63
Quick Amp Labeling Kit Sample Preparation 64
Step 1. Prepare Spike Mix 66
Step 2. Prepare labeling reaction 69
Step 3. Purify the labeled/amplified RNA 72
Step 4. Quantify the cRNA 72
Quality Assessment of Template RNA and Labeled cRNA 73
Step 1. Prepare for quality assessment 74
Step 2. Assess the quality using the Agilent 2100 Bioanalyzer 75
Step 3. Assess the quality using a NanoDrop Spectrophotometer 78
Preventing Ozone-Related Problems 79
Step 1. Prepare the Stabilization and Drying Solution 80
Step 2. Wash with Stabilization and Drying Solution 81
Normalizing Agilent One-Color Microarray Data 84
To do downstream analysis of Agilent microarray data 84
To use Feature Extraction 85

4 Reference 87
Kit Contents 88
Supplemental User Guides 91
Microarray Handling Tips 92
General Microarray Layout and Orientation 93
Array/Sample tracking microarray slides 96
Related Microarray Reagents 98

6 One-Color Microarray-Based Gene Expression Analysis (Low Input Quick Amp Labeling) Protocol
 One-Color Microarray-Based Gene Expression Analysis
Protocol

1
Before You Begin
Procedural Notes 8
Safety Notes 9
Agilent Oligo Microarrays 10
Required Equipment 13
Required Reagents 15
Optional Equipment/Reagents 16
Required Hardware and Software 16
Optional Software 17

Make sure you read and understand the information in this chapter and have
the necessary equipment and reagents listed before you start an experiment.

Agilent cannot guarantee microarray performance and does not provide technical support
NOTE to those who use non-Agilent protocols in processing Agilent microarrays.

7
1 Before You Begin 
Procedural Notes

Procedural Notes
• Determine the integrity and purity of the input RNA for labeling and
hybridization prior to use to increase the likelihood of a successful
experiment.
• To prevent contamination of reagents by nucleases, always wear
powder-free laboratory gloves, and use dedicated solutions and pipettors
with nuclease-free aerosol-resistant tips.
• Maintain a clean work area.
• When preparing frozen reagent stock solutions for use:
1 Thaw the aliquot as rapidly as possible without heating above room
temperature, unless otherwise indicated.
2 Mix briefly on a vortex mixer, then spin in a centrifuge for 5 to
10 seconds to drive the contents off of walls and lid.
3 Store on ice or in a cold block until use, unless otherwise indicated.
• In general, follow Biosafety Level 1 (BL1) safety rules.

8 One-Color Microarray-Based Gene Expression Analysis (Low Input Quick Amp Labeling) Protocol
 Before You Begin 1
Safety Notes

Safety Notes
• Inspect the Stabilization and Drying Solution bottle for chips or cracks prior to use.
CAUTION
Failure to do so may result in bottle breakage.
• Wear appropriate personal protective equipment (PPE) when working in the
laboratory.

• Cyanine dye reagents are potential carcinogens. Avoid inhalation, swallowing,

WA R N I N G
or contact with skin.
• LiCl is toxic and a potential teratogen. May cause harm to breastfed babies.
Possible risk of impaired fertility. Harmful if inhaled, swallowed, or contacts
skin. Target organ: central nervous system. Wear suitable PPE. LiCl is a
component of the 2× Hi-RPM Hybridization Buffer.
• Lithium dodecyl sulfate (LDS) is harmful by inhalation and irritating to eyes,
respiratory system, and skin. Wear suitable PPE. LDS is a component of the 2×
Hi-RPM Hybridization Buffer.
• Triton is harmful if swallowed. Risk of serious damage to eyes. Wear suitable
PPE. Triton is a component of the 2× Hi-RPM Hybridization Buffer and is an
additive in wash buffers.
• Acetonitrile is a flammable liquid and vapor. Harmful if inhaled, swallowed, or
contacts skin. Target organs: liver, kidneys, cardiovascular system, and CNS.
• Stabilization and Drying Solution is toxic and flammable and must be used in a
suitable fume hood. This solution contains acetonitrile and must be disposed of
in a manner consistent with disposal of like solvents. Gloves and eye/face
protection should be used during every step of this protocol, especially when
handling acetonitrile and the Stabilization and Drying Solution.

One-Color Microarray-Based Gene Expression Analysis (Low Input Quick Amp Labeling) Protocol 9
1 Before You Begin 
Agilent Oligo Microarrays

Agilent Oligo Microarrays

For more information on microarray designs visit the following web site:
[Link]
To get design files or create a custom design, go to the Agilent eArray web site
at [Link]

Store entire kit at room temperature. After breaking foil on microarray pouch, store
NOTE microarray slides at room temperature (in the dark) under a vacuum dessicator or nitrogen
purge box. Do not store microarray slides in open air after breaking foil.

Two, four or eight microarrays printed on each 1-inch × 3-inch glass slide

Catalog SurePrint HD and G3 Microarrays and Microarray Kits

Table 1 Catalog SurePrint G3 and HD Microarrays - Human

Part Number Description

G4851C G3 Human Gene Expression 8×60K v3 Microarray Kit (3 slides)
G4858A-072363 G3 Human Gene Expression 8×60K v3 Microarray (1 slide)
G4851B G3 Human Gene Expression 8×60K v2 Microarray Kit (3 slides)
G4858A-039494 G3 Human Gene Expression 8×60K v2 Microarray (1 slide)
G4851A G3 Human GE 8×60K Microarray Kit (3 slides)
G4858A-028004 G3 Human GE 8×60K Microarray (1 slide)
G4845A HD Human GE 4×44K v2 Microarray Kit (5 slides)
G2519F-026652 HD Human GE 4×44K v2 Microarray (1 slide)

10 One-Color Microarray-Based Gene Expression Analysis (Low Input Quick Amp Labeling) Protocol
 Before You Begin 1
Agilent Oligo Microarrays

Table 2 Catalog SurePrint G3 and HD Microarrays - Mouse

Part Number Description

G4852B G3 Mouse GE 8×60K v2 Microarray Kit (3 slides)
G4858A-074809 G3 Mouse GE 8×60K v2 Microarray (1 slide)
G4852A G3 Mouse GE 8×60K Microarray Kit (3 slides)
G4858A-028005 G3 Mouse GE 8×60K Microarray (1 slide)
G4846A HD Mouse GE 4×44K v2 Microarray Kit (5-slides)
G2519F-026655 HD Mouse GE 4×44K v2 Microarray (1 slide)
G4122F HD Whole Mouse Genome Microarray Kit, 4×44K (5 slides)
G2519F-014868 HD Whole Mouse Genome Microarray Kit, 4×44K (1 slide)

Table 3 Catalog SurePrint G3 and HD Microarrays - Rat

Part Number Description

G4853B G3 Rat GE 8×60K v2 Microarray Kit (3 slides)
G4858A-074036 G3 Rat GE 8×60K v2 Microarray (1 slide)
G4853A G3 Rat GE 8×60K Microarray Kit (3 slides)
G4858A-028279 G3 Rat GE 8×60K Microarray (1 slide)
G4847B HD Rat GE 4×44K v3 Microarray Kit (3 slides)
G2519F-028282 HD Rat GE 4×44K v3 Microarray (1 slide)

One-Color Microarray-Based Gene Expression Analysis (Low Input Quick Amp Labeling) Protocol 11
1 Before You Begin 
Agilent Oligo Microarrays

Table 4 Catalog SurePrint HD Microarrays and Microarray Kits - Model

Organisms/Non-Human

Part Number Description

G2519F-021169 Arabidopsis (V4) Gene Expression Microarray, 4×44K (1 slide)
G2519F-021623 Barley Gene Expression Microarray, 4×44K (1 slide)
G2519F-023647 Bovine (V2) Gene Expression Microarray, 4×44K (1 slide)
G2519F-022520 Brassica Gene Expression Microarray, 4x44K (1 slide)
G2519F-021193 Canine (V2) Gene Expression Microarray, 4×44K (1 slide)
G2519F-020186 C. elegans (V2) Gene Expression Microarray, 4×44K (1 slide)
G2519F-026441 Chicken (V2) Gene Expression Microarray, 4×44K (1 slide)
G2519F-022523 Cotton Gene Expression Microarray, 4×44K (1 slide)
G2519F-021791 Drosophila Gene Expression Microarray, 4×44K (1 slide)
G2519F-021322 Horse Gene Expression Microarray, 4×44K (1 slide)
G2519F-015060 Magnaporthe (V2) Gene Expression Microarray, 4×44K (1 slide)
G2519F-022524 Medicago Gene Expression Microarray, 4×44K (1 slide)
G2519F-020449 Mosquito Gene Expression Microarray, 4×44K (1 slide)
G2519F-026440 Porcine (V2) Gene Expression Microarray, 4×44K (1 slide)
G2519F-026806 Rhesus Macaque (V2) Gene Expression Microarray, 4×44K (1 slide)
G2519F-020908 Rabbit Gene Expression Microarray, 4×44K (1 slide)
G2519F-015241 Rice Gene Expression Microarray, 4×44K (1 slide)
G2519F-020938 Salmon Gene Expression Microarray, 4×44K (1 slide)
G4813A-019921 Sheep Gene Expression Microarray, 8×15K (1 slide)
G2519F-021113 Tobacco Gene Expression Microarray, 4×44K (1 slide)
G2519F-022297 Wheat Gene Expression Microarray, 4×44K (1 slide)
G4813A-016322 Yeast (V2) Gene Expression Microarray, 8×15K (1 slide)
G2519F-026437 Zebrafish (V3) Gene Expression Microarray, 4×44K (1 slide)

12 One-Color Microarray-Based Gene Expression Analysis (Low Input Quick Amp Labeling) Protocol
 Before You Begin 1
Required Equipment

Custom Microarrays
One, two, four, or eight microarrays printed on each 1-inch × 3-inch glass slide.

Table 5 Custom SurePrint HD Microarrays

Part Number Description

G4502A Custom Gene Expression Microarray, 1×244K
G4503A Custom Gene Expression Microarray, 2×105K
G2514F Custom Gene Expression Microarray, 4×44K
G2509F Custom Gene Expression Microarray, 8×15K

Table 6 Custom SurePrint G3 Microarrays

Part Number Description

G4860A SurePrint G3 Custom Gene Expression Microarray, 1×1M
G4861A SurePrint G3 Custom Gene Expression Microarray, 2×400K
G4862A SurePrint G3 Custom Gene Expression Microarray, 4×180K
G4102A SurePrint G3 Custom Gene Expression Microarray, 8×60K

Required Equipment
Table 8 Required Equipment

Description Vendor and part number

Agilent Microarray Scanner Agilent p/n G4900DA, G2565CA or G2565BA
Hybridization Chamber, stainless Agilent p/n G2534A
Hybridization gasket slides 
1 microarray/slide, 5 slides/box Agilent p/n G2534-60003
2 microarrays/slide, 5 slides/box Agilent p/n G2534-60002
4 microarrays/slide, 5 slides/box Agilent p/n G2534-60011
8 microarrays/slide, 5 slides/box Agilent p/n G2534-60014
Go to [Link]/genomics to see all
available kit configurations.

One-Color Microarray-Based Gene Expression Analysis (Low Input Quick Amp Labeling) Protocol 13
1 Before You Begin 
Required Equipment

Table 8 Required Equipment (continued)

Description Vendor and part number

Hybridization oven; temperature set at 65°C Agilent p/n G2545A
Hybridization oven rotator for Agilent Agilent p/n G2530-60029
Microarray Hybridization Chambers
nuclease-free 1.5 mL microfuge tube Ambion p/n 12400 or equivalent
magnetic stir bar (×2) Corning p/n 401435 or equivalent
magnetic stir plate (×2) Corning p/n 6795-410 or equivalent
circulating water baths or heat blocks set to Corning p/n 6795-420 or equivalent
37°C, 40°C, 60°C, 65°C, 70°C, and 80°C,
microcentrifuge Eppendorf p/n 5417R or equivalent
sterile storage bottle Nalgene 455-1000 or equivalent
spectrophotometer NanoDrop p/n ND-1000 UV-VIS or equivalent
micropipettor Pipetman P-10, P-20, P-200, P-1000 or
equivalent
slide-staining dish, with slide rack (×3) Thermo Shandon p/n 121 or equivalent
clean forceps
ice bucket
powder-free gloves
sterile, nuclease-free aerosol barrier pipette tips
vortex mixer
timer
nitrogen purge box for slide storage

14 One-Color Microarray-Based Gene Expression Analysis (Low Input Quick Amp Labeling) Protocol
 Before You Begin 1
Required Reagents

Required Reagents
Table 9 Required Reagents

Description Vendor and part or catalog number

Low Input Quick Amp Labeling Kit, One-Color Agilent p/n 5190-2305
RNA Spike-In Kit, One-Color Agilent p/n 5188-5282
Gene Expression Hybridization Kit Agilent p/n 5188-5242
Gene Expression Wash Buffer Kit Agilent p/n 5188-5327
DNase/RNase-free distilled water Invitrogen p/n 10977-015
RNeasy Mini Kit Qiagen p/n 74104
ethanol (95% to 100% molecular biology grade) Sigma-Aldrich p/n E7023-6×500ML
Milli-Q water or equivalent
isopropyl alcohol (molecular biology grade)

One-Color Microarray-Based Gene Expression Analysis (Low Input Quick Amp Labeling) Protocol 15
1 Before You Begin 
Optional Equipment/Reagents

Optional Equipment/Reagents
Table 10 Optional Equipment/Reagents

Description Vendor and part number

2100 Bioanalyzer Agilent p/n G2939AA
RNA 6000 Nano Assay Kit (RNA Series II Kit) Agilent p/n 5067-1511
Quick Amp Labeling Kit, One-Color Agilent p/n 5190-0442
*
Stabilization and Drying Solution Agilent p/n 5185-5979
Ozone-Barrier Slide Cover* Agilent p/n G2505-60550
Absolutely RNA Nanoprep Kit Agilent p/n 400753
slide box Corning p/n 07201629
acetonitrile Sigma p/n 271004-1L
sulfolane Sigma p/n T22209
thermal cycler
PCR 96-well plate or 0.2 mL PCR tubes

* Recommended when processing microarrays in high ozone environment.

Required Hardware and Software

Table 11

Description
Feature Extraction software 10.7.1 or later
Agilent Scan Control software. Refer to Agilent Scanner user guide for specifications.
For system and supported Internet Explorer/Adobe Reader versions, please see the System
Requirements for your Feature Extraction and Scan Control Software.

16 One-Color Microarray-Based Gene Expression Analysis (Low Input Quick Amp Labeling) Protocol
 Before You Begin 1
Optional Software

Optional Software
Table 12

Description
GeneSpring GX 9.0 or later

One-Color Microarray-Based Gene Expression Analysis (Low Input Quick Amp Labeling) Protocol 17
 This page intentionally left blank.

18 One-Color Microarray-Based Gene Expression Analysis (Low Input Quick Amp Labeling) Protocol
 One-Color Microarray-Based Gene Expression Analysis
Protocol

2
Procedures
Sample Preparation 21
Step 1. Prepare Spike Mix 23
Step 2. Prepare labeling reaction 26
Step 3. Purify the labeled/amplified RNA 30
Step 4. Quantify the cRNA 32
Hybridization 34
Step 1. Prepare the 10× Blocking Agent 34
Step 2. Prepare hybridization samples 35
Step 3. Prepare the hybridization assembly 37
Microarray Wash 40
Step 1. Add Triton X-102 to Gene Expression wash buffers 40
Step 2. Prewarm Gene Expression Wash Buffer 2 41
Step 3. Prepare the equipment 41
Step 4. Wash the microarray slides 43
Scanning and Feature Extraction 47
Step 1. Scan the slides 47
Step 2. Extract data using Agilent Feature Extraction Software 50

The Agilent One-Color Microarray-based Gene Expression Analysis uses

cyanine 3-labeled targets to measure gene expression in experimental and
control samples. Figure 1 is a standard workflow for sample preparation and
array hybridization design.

19
2 Procedures 

Template Total or poly A+ RNA with Spike-In

cDNA synthesis*

cRNA synthesis and amplification*

cRNA purification*

Preparation of hybridization sample

17-hour hybridization (65ºC)

Wash

Scan

Feature Extraction

* Samples can be stored frozen at -80ºC after these steps, if needed.

Figure 1 Workflow for sample preparation and array processing.

20 One-Color Microarray-Based Gene Expression Analysis (Low Input Quick Amp Labeling) Protocol
 Procedures 2
Sample Preparation

Sample Preparation
The Low Input Quick Amp Labeling Kit, One-Color generates fluorescent cRNA
(complimentary RNA) with a sample input RNA range between 10 ng and
200 ng of total RNA or a minimum of 5 ng of poly A+ RNA for one-color
processing. The method uses T7 RNA Polymerase Blend (red cap), which
simultaneously amplifies target material and incorporates Cyanine 3-CTP.
Amplification is typically at least a 100-fold from total RNA to cRNA with the
use of this kit.

For optimal performance, use pure high quality, intact template total or poly A+ RNA. RNA
NOTE that is not pure, as measured by A260/A230 ratio, can lead to poor results and must be
purified. Please refer to “Quality Assessment of Template RNA and Labeled cRNA” on
page 73 for general guidance and procedural recommendations on quality assessment of
template RNA.

One-Color Microarray-Based Gene Expression Analysis (Low Input Quick Amp Labeling) Protocol 21
2 Procedures 
Sample Preparation

Figure 2 Schematic of amplified cRNA procedure. Generation of cRNA for a two-color

microarray experiment is shown.

22 One-Color Microarray-Based Gene Expression Analysis (Low Input Quick Amp Labeling) Protocol
 Procedures 2
Step 1. Prepare Spike Mix

Step 1. Prepare Spike Mix

(Time required: ~0.5 hours)
Refer to the protocol for RNA Spike-In Kit, One-Color for in-depth instructions
and troubleshooting advice on how to use one-color spike mixes. This protocol
is available with the RNA Spike-In Kit, One-Color and can also be downloaded
from the Agilent web site at [Link]/chem/dnamanuals-protocols.
1 Equilibrate water baths to 37°C, 40°C, 65°C, 70°C, and 80°C.
2 Vigorously mix the Spike Mix solution on a vortex mixer.
3 Heat at 37°C for 5 minutes, and mix on a vortex mixer once more.
4 Briefly spin in a centrifuge to drive contents to the bottom of the tube prior
to opening. Settlement of the solution on the sides or lid of the tubes may
occur during shipment and storage.
Table 13 provides the dilutions of Spike Mix for a range of total RNA input
amounts. For inputs not shown Table 13, make sure that the amount of spike
mix is proportional to the amount of RNA input. If you start with 5 ng mRNA
as the input mass, follow the dilution scheme as described in Table 13.

Table 13 Dilutions of Spike Mix for Cyanine 3-labeling

Starting Amount of RNA Serial Dilution Spike Mix Volume to be used

in each labeling reaction (µL)
Total PolyA First Second Third Fourth
RNA (ng) RNA (ng)

10 1:20 1:25 1:20 1:10 2

25 1:20 1:25 1:20 1:4 2

50 1:20 1:25 1:20 1:2 2

100 1:20 1:25 1:20 2

200 1:20 1:25 1:10 2

5 1:20 1:25 1:20 2

Use RNase-free microfuge tubes and tips. Make sure you dispense at least 2 µL with a
NOTE pipette to ensure accuracy.

One-Color Microarray-Based Gene Expression Analysis (Low Input Quick Amp Labeling) Protocol 23
2 Procedures 
Step 1. Prepare Spike Mix

For example, to prepare the Spike Mix dilution appropriate for 25 ng of total
RNA starting sample:
1 Create the First Dilution:
a Label a new sterile 1.5-mL microcentrifuge tube “Spike Mix First
Dilution.”
b Mix the thawed Spike Mix vigorously on a vortex mixer.
c Heat at 37°C in a circulating water bath for 5 minutes.
d Mix the Spike Mix tube vigorously again on a vortex mixer.
e Spin briefly in a centrifuge to drive contents to the bottom of the tube.
f Into the First Dilution tube, put 2 μL of Spike Mix stock.
g Add 38 μL of Dilution Buffer provided in the Spike-In kit (1:20).
h Mix thoroughly on a vortex mixer and spin down quickly in a
microcentrifuge to collect all of the liquid at the bottom of the tube. This
tube contains the First Dilution.
2 Create the Second Dilution:
a Label a new sterile 1.5-mL microcentrifuge tube “Spike Mix Second
Dilution.”
b Into the Second Dilution tube, put 2 μL of First Dilution.
c Add 48 μL of Dilution Buffer (1:25).
d Mix thoroughly on a vortex mixer and spin down quickly in a
microcentrifuge to collect all of the liquid at the bottom of the tube. This
tube contains the Second Dilution.
3 Create the Third Dilution:
a Label a new sterile 1.5-mL microcentrifuge tube “Spike Mix Third
Dilution.”
b Into the Third Dilution tube, put 2 μL of Second Dilution.
c Add 38 μL of Dilution Buffer (1:20).
d Mix thoroughly on a vortex mixer and spin down quickly in a
microcentrifuge to collect all the liquid at the bottom of the tube. This
tube contains the Third Dilution.

24 One-Color Microarray-Based Gene Expression Analysis (Low Input Quick Amp Labeling) Protocol
 Procedures 2
Step 1. Prepare Spike Mix

4 Create the Fourth Dilution:

a Label a new sterile 1.5-mL microcentrifuge tube “Spike Mix Fourth
Dilution.”
b Into the Fourth Dilution tube, add 10 μL of Third Dilution to 30 μL of
Dilution Buffer for the Fourth Dilution (1:4).
c Mix thoroughly on a vortex mixer and spin down quickly in a
microcentrifuge to collect all of the liquid at the bottom of the tube. This
tube contains the Fourth Dilution (now at a 40,000-fold final dilution).
5 Add 2 μL of Fourth Dilution to 25 ng of sample total RNA as listed in
Table 13 and continue with cyanine 3 labeling using the Agilent Low Input
Quick Amp Kit protocol as described in “Step 2. Prepare labeling
reaction” on page 26.

Storage of Spike Mix dilutions

Store the RNA Spike-In Kit, One-Color at –70°C to –80°C in a non-defrosting
freezer for up to 1 year from the date of receipt.
Store the first dilution of the Spike Mix positive controls for up to 2 months in
a non-defrosting freezer at -70°C to -80°C. Do not freeze/thaw more than eight
times. After use, discard the second, third and fourth dilution tubes.

One-Color Microarray-Based Gene Expression Analysis (Low Input Quick Amp Labeling) Protocol 25
2 Procedures 
Step 2. Prepare labeling reaction

Step 2. Prepare labeling reaction

(Time required: ~5.5 hours)
For each assay, make sure that the volume of the total RNA sample plus
diluted RNA spike-in controls does not exceed 3.5 μL. Because the 1× reaction
involves volumes of less than 1 μL, prepare components in a master mix and
divide into the individual assay tubes in volumes >1 μL. When preparing 4
samples, use the 5× master mix. When preparing 8 samples, use the 10×
master mix.

The starting input for the Low Input Quick Amp Labeling Kit, One-Color ranges from 10 ng
NOTE to 200 ng of total RNA. For best results, start with at least 25 ng of total RNA for the
4-pack and 8-pack formats, and 50 ng of total RNA for the 1-pack and 2-pack formats. For
the 8-pack microarray format, as little as 10 ng of total RNA can be used to generate high
quality data.

1 Add 10 to 200 ng of total RNA to a 1.5-mL microcentrifuge tube in a final

volume of 1.5 μL. If samples are concentrated, dilute with water until 10 to
200 ng of total RNA is added in a 1.5 μL volume. Dilute the total RNA just
prior to use and store the total RNA at concentrations over 100 ng/μL.
2 Add 2 μL of diluted Spike Mix to each tube. Each tube now contains a total
volume of 3.5 μL.

26 One-Color Microarray-Based Gene Expression Analysis (Low Input Quick Amp Labeling) Protocol
 Procedures 2
Step 2. Prepare labeling reaction

3 Prepare T7 Primer Mix and add to sample:

a Mix T7 Primer (green cap) and water as listed in Table 14.

Table 14 T7 Primer Mix

Component Volume (µL) per Volume (µL) per 5 Volume (µL) per
reaction reaction 10 reactions

T7 Primer (green cap) 0.8 4 8

Nuclease-free Water 1 5 10

Total Volume 1.8 9 18

b Add 1.8 μL of T7 Primer Mix into each tube that contain 3.5 μL of total
RNA and diluted RNA spike-in controls. Each tube now contains a total
volume of 5.3 μL.
c Denature the primer and the template by incubating the reaction at 65°C
in a circulating water bath for 10 minutes.
d Put the reactions on ice and incubate for 5 minutes.
4 Prewarm the 5× First Strand Buffer (green cap) at 80°C for 3 to 4 minutes
to ensure adequate resuspensions of the buffer components. For optimal
resuspension, briefly mix on a vortex mixer and spin the tube in a
microcentrifuge to drive down the contents from the tube walls. Keep at
room temperature until needed.
5 Prepare and add cDNA Master Mix:
a Immediately prior to use, add the components in Table 15 to a 1.5-mL
microcentrifuge tube. Use a pipette to gently mix. Keep at room
temperature.
The Affinity Script RNase Block Mix (violet cap) is a blend of enzymes.
Keep the Affinity Script RNase Block Mix (violet cap) on ice and add to
the cDNA Master Mix immediately prior to use.

One-Color Microarray-Based Gene Expression Analysis (Low Input Quick Amp Labeling) Protocol 27
2 Procedures 
Step 2. Prepare labeling reaction

Table 15 cDNA Master Mix

Component Volume (µL) Volume (µL) Volume (µL)

per reaction per 5 reaction per 10
reactions

5× First Strand Buffer (green cap) 2 10 20

0.1 M DTT (white cap) 1 5 10

10 mM dNTP Mix (green cap) 0.5 2.5 5

Affinity Script RNase Block Mix (violet 1.2 6 12

cap)
Total Volume 4.7 23.5 47

b Briefly spin each sample tube in a microcentrifuge to drive down the

contents from the tube walls and the lid.
c Add 4.7 μL of cDNA Master Mix to each sample tube and mix by pipetting
up and down. Each tube now contains a total volume of 10 μL.
d Incubate samples at 40°C in a circulating water bath for 2 hours.
e Move samples to a 70°C circulating water bath and incubate for
15 minutes.

Incubation at 70°C inactivates the AffinityScript enzyme.

NOTE

f Move samples to ice. Incubate for 5 minutes.

g Spin samples briefly in a microcentrifuge to drive down tube contents
from the tube walls and lid.
Stopping Point If you do not immediately continue to the next step, store the samples at
-80°C.

6 Prepare and add Transcription Master Mix:

a Immediately prior to use, add the components in Table 16 in the order
listed into a 1.5 mL microcentrifuge tube. Use a pipette to gently mix.
Keep at room temperature.
The T7 RNA Polymerase Blend (red cap) is a blend of enzymes. Keep the
T7 RNA Polymerase Blend (red cap) on ice and add to the Transcription
Master Mix just before use.

28 One-Color Microarray-Based Gene Expression Analysis (Low Input Quick Amp Labeling) Protocol
 Procedures 2
Step 2. Prepare labeling reaction

Table 16 Transcription Master Mix

Component Volume (µL) per Volume (µL) per Volume (µL)

reaction 5 reaction per 10
reactions

Nuclease-free water (white cap) 0.75 3.75 7.5

5× Transcription Buffer (blue cap) 3.2 16 32

0.1 M DTT (white cap) 0.6 3 6

NTP Mix (blue cap) 1 5 10

T7 RNA Polymerase Blend (red cap) 0.21 1.05 2.1

Cyanine 3-CTP 0.24 1.2 2.4

Total Volume 6 30 60

b Add 6 μL of Transcription Master Mix to each sample tube. Gently mix by

pipetting. Each tube now contains a total volume of 16 μL.
c Incubate samples in a circulating water bath at 40°C for 2 hours.
Stopping Point If you do not immediately continue to the next step, store the samples at
-80°C.

One-Color Microarray-Based Gene Expression Analysis (Low Input Quick Amp Labeling) Protocol 29
2 Procedures 
Step 3. Purify the labeled/amplified RNA

Step 3. Purify the labeled/amplified RNA

(Time required: ~0.5 hours)
Use the RNeasy Mini Kit to purify the amplified cRNA samples.
If sample concentration causes difficulty, you can use the Absolutely RNA
Nanoprep Kit as an alternative. See “Absolutely RNA Nanoprep
Purification” on page 58.

Make sure that ethanol was added to the RPE buffer as specified in the Qiagen manual
NOTE before you continue.

1 Add 84 μL of nuclease-free water to your cRNA sample, for a total volume of

100 μL.
2 Add 350 μL of Buffer RLT and mix well by pipetting.
3 Add 250 μL of ethanol (96% to 100% purity) and mix thoroughly by
pipetting. Do not spin in a centrifuge.
4 Transfer the 700 μL of the cRNA sample to an RNeasy Mini Spin Column
(pink) in a Collection Tube (2 ml). Spin the sample in a centrifuge at 4°C for
30 seconds at 13,000 rpm. Discard the flow-through and collection tube.
5 Transfer the RNeasy column to a new Collection Tube (2 ml) and add
500 μL of Buffer RPE (containing ethanol) to the column. Spin the sample
in a centrifuge at 4°C for 30 seconds at 13,000 rpm. Discard the
flow-through. Re-use the collection tube.
6 Add another 500 μL of Buffer RPE to the column. Centrifuge the sample at
4°C for 60 seconds at 13,000 rpm. Discard the flow-through and the
collection tube.
7 If any Buffer RPE remains on or near the frit of the column or on the
outside of the column, transfer the RNeasy column to a new Collection Tube
(1.5 ml) and spin the sample in a centrifuge at 4°C for 30 seconds at
13,000 rpm to remove any remaining traces of Buffer RPE. Discard this
collection tube and use a fresh Collection Tube (1.5 ml) to elute the cleaned
cRNA sample.

Do not discard the final flow-through in the next step. It contains the cRNA sample.
CAUTION

30 One-Color Microarray-Based Gene Expression Analysis (Low Input Quick Amp Labeling) Protocol
 Procedures 2
Step 3. Purify the labeled/amplified RNA

8 Elute the purified cRNA sample by transferring the RNeasy column to a

new Collection Tube (1.5 ml). Add 30 μL RNase-Free Water directly onto the
RNeasy filter membrane. Wait 60 seconds, then centrifuge at 4°C for
30 seconds at 13,000 rpm.
9 Maintain the cRNA sample-containing flow-through on ice. Discard the
RNeasy column.

One-Color Microarray-Based Gene Expression Analysis (Low Input Quick Amp Labeling) Protocol 31
2 Procedures 
Step 4. Quantify the cRNA

Step 4. Quantify the cRNA

Use the NanoDrop ND-1000 UV-VIS Spectrophotometer version 3.2.1 (or
higher) to quantify the cRNA.

1 Start the NanoDrop software.

2 Click the Microarray Measurement tab.
3 Before initializing the instrument as requested by the software, clean the
sample loading area with nuclease-free water.
4 Load 1.0 to 2.0 μL of nuclease-free water to initialize. Then click OK.
5 Once the instrument has initialized, select RNA-40 as the Sample type (use
the drop down menu).
6 Make sure the Recording button is selected. If not, click Recording so that
the readings can be recorded, saved, and printed.

Failure to engage recording causes measurements to be overwritten, with no

CAUTION
possibility of retrieval.

7 Blank the instrument by pipetting 1.0 to 2.0 μL of nuclease-free water (this

can be the same water used to initialize the instrument) and click Blank.
8 Clean the sample loading area with a laboratory wipe. Pipette 1.0 to 2.0 μL
of the sample onto the instrument sample loading area. Type the sample
name in the space provided and click Measure.
Be sure to clean the sample loading area between measurements and
ensure that the baseline is always flat at 0, which is indicated by a thick
black horizontal line. If the baseline deviates from 0 and is no longer a flat
horizontal line, reblank the instrument with nuclease-free water, then
remeasure the sample.
9 Print the results. If printing the results is not possible, record the following
values:
• Cyanine 3 dye concentration (pmol/μL)
• RNA absorbance ratio (260 nm/280 nm)
• cRNA concentration (ng/μL)

32 One-Color Microarray-Based Gene Expression Analysis (Low Input Quick Amp Labeling) Protocol
 Procedures 2
Step 4. Quantify the cRNA

10 Determine the yield and specific activity of each reaction as follows:

a Use the concentration of cRNA (ng/μL) to determine the μg cRNA yield
as follows:

(Concentration of cRNA)  30 μL (elution volume)

----------------------------------------------------------------------------------------------------------------------------------------- = μg of cRNA
1000
b Use the concentrations of cRNA (ng/μL) and cyanine 3 (pmol/μL) to
determine the specific activity as follows:

Concentration of Cy3
--------------------------------------------------------------  1000 = pmol Cy3 per μg cRNA
Concentration of cRNA
11 Examine the yield and specific activity results. See Table 17 for the
recommended cRNA yields and specific activities for hybridization.

If the specific activity does not meet the requirements listed in Table 17, do not
CAUTION
continue to hybridization. Repeat preparation of cRNA.

Table 17 Recommended Yields and Specific Activity

Microarray format Yield (µg) Specific Activity (pmol Cy3 per µg cRNA)

1-pack 5 6

2-pack 3.75 6

4-pack 1.65 6

8-pack 0.825 6

Please refer to “Quality Assessment of Template RNA and Labeled cRNA” on page 73 for
NOTE general guidance and procedural recommendations on quality assessment of labeled
cRNA.

One-Color Microarray-Based Gene Expression Analysis (Low Input Quick Amp Labeling) Protocol 33
2 Procedures 
Hybridization

Hybridization
An instructional video that shows hybridization and washing steps can be
found at [Link] Search for “Running a microarray
experiment”.
If you are a first time user, practice the hybridization process before you begin.
Use water instead of blocking mix, and use a clean microscope slide and a
gasket slide. Make sure you mix and apply the hybridization solution with
minimal bubbles. Practice the hyb assembly and the slide disassembly and
wash.

You must calibrate the hybridization oven regularly for accuracy of the collected data.
CAUTION
Refer to Agilent G2545A Hybridization Calibration Procedure (p/n G2545-90002,
version A1 or higher) for more information.

Step 1. Prepare the 10× Blocking Agent

1 Add 500 μL of nuclease-free water to the vial containing lyophilized 10×
Gene Expression Blocking Agent supplied with the Gene Expression
Hybridization Kit, or add 1250 μL of nuclease-free water to the vial
containing lyophilized large volume 10× Gene Expression Blocking Agent.
2 Gently mix on a vortex mixer. If the pellet does not go into solution
completely, heat the mix for 4 to 5 minutes at 37°C.
3 Drive down any material that sticks to the tube walls or cap by spinning in a
centrifuge for 5 to 10 seconds.

Divide the 10× Gene Expression Blocking Agent into aliquots small enough to keep the
NOTE freeze-thaw cycle to 5 times or less. Store at -20°C for up to two months. Before use,
repeat step 2 and step 3.

34 One-Color Microarray-Based Gene Expression Analysis (Low Input Quick Amp Labeling) Protocol
 Procedures 2
Step 2. Prepare hybridization samples

Step 2. Prepare hybridization samples

1 Equilibrate water bath to 60°C.
2 For each microarray, add each of the components as indicated in Table 18
or Table 19 to a 1.5 mL nuclease-free microfuge tube:
3 Mix well but gently on a vortex mixer.

For 1-pack and 2-pack microarrays, if you did not generate enough labeled cRNA, add the
NOTE amount of labeled cRNA to the fragmentation mix such that the same amount is used for
each microarray within the same experiment (at least 1.65 µg).

Table 18 Fragmentation mix for 1-pack or 2-pack microarray formats

Components Volume/Mass Volume/Mass

1-pack microarrays 2-pack microarrays
Cyanine 3-labeled, linearly amplified cRNA 5.00 µg 3.75 µg
10× Gene Expression Blocking Agent 50 µL 25 µL
Nuclease-free water bring volume to 240 µL bring volume to 120 µL
25× Fragmentation Buffer 10 µL 5 µL
Total Volume 250 µL 125 µL

Table 19 Fragmentation mix for 4-pack or 8-pack microarray formats

Components Volume/Mass Volume/Mass

4-pack microarrays 8-pack microarrays
Cyanine 3-labeled, linearly amplified cRNA 1.65 µg 600 ng
10× Gene Expression Blocking Agent 11 µL 5 µL
Nuclease-free water bring volume to 52.8 µL bring volume to 24 µL
25× Fragmentation Buffer 2.2 µL 1 µL
Total Volume 55 µL 25 µL

Do not incubate sample in the next step for more than 30 minutes. Cooling on ice and
CAUTION
adding the 2× Hi-RPM Hybridization Buffer stops the fragmentation reaction.

4 Incubate at 60°C for exactly 30 minutes to fragment RNA.

One-Color Microarray-Based Gene Expression Analysis (Low Input Quick Amp Labeling) Protocol 35
2 Procedures 
Step 2. Prepare hybridization samples

5 Immediately cool on ice for one minute.

6 Add 2× Hi-RPM Hybridization Buffer to stop the fragmentation reaction.
See Table 20.

Table 20 Hybridization mix

Volumes per hybridization

Components 1-pack 2-pack 4-pack 8-pack
cRNA from Fragmentation Mix 250 µL 125 µL 55 µL 25 µL
2× Hi-RPM Hybridization Buffer 250 µL 125 µL 55 µL 25 µL

7 Mix well by careful pipetting part way up and down. Do not introduce
bubbles to the mix. The surfactant in the 2× Hi-RPM Hybridization Buffer
easily forms bubbles. Do not mix on a vortex mixer; mixing on a vortex
mixer introduces bubbles.
8 Spin for 1 minute at room temperature at 13,000 rpm in a microcentrifuge
to drive the sample off the walls and lid and to aid in bubble reduction.
Use immediately. Do not store.
9 Put sample on ice and load onto the array as soon as possible.
Refer to “Microarray Handling Tips” on page 92 for information on how to
safely handle microarrays.

36 One-Color Microarray-Based Gene Expression Analysis (Low Input Quick Amp Labeling) Protocol
 Procedures 2
Step 3. Prepare the hybridization assembly

Step 3. Prepare the hybridization assembly

Refer to the Agilent Microarray Hybridization Chamber User Guide for more
details to load slides, and to assemble and disassemble the chambers. This
user guide is included with the Agilent Microarray Hybridization Chamber Kit
(G2534A) and can also be downloaded from the Agilent web site at
[Link]. Search for G2534A.

1 Load a clean gasket slide into the Agilent SureHyb chamber base with the
label facing up and aligned with the rectangular section of the chamber
base. Make sure that the gasket slide is flush with the chamber base and is
not ajar.

Do not let the pipette tip or the hybridization solution touch the gasket walls. Allowing
CAUTION
liquid to touch the gasket wall greatly increases the likelihood of gasket leakage.
When you lower the microarray slide on top of the SureHyb gasket slide, make sure
that the two slides are parallel at all times.

2 Slowly dispense the volume of hybridization sample (see Table 21) onto the
gasket well in a “drag and dispense” manner.
• Position the slides so that the barcode label is to your left.
• Load the samples left to right. For 8-pack slides, start with the first row.
The output files will come out in that same order. Refer to “Array/Sample
tracking microarray slides” on page 96 for guidelines on tracking sample
position for multipack slide formats.
• Avoid the introduction of air bubbles to the gasket wells. Air bubbles can
affect the final sample volume and can cause leakage from the gasket
well.

Table 21 Hybridization Sample

Volumes per hybridization

Components 1-pack 2-pack 4-pack 8-pack
Volume Prepared 500 µL 250 µL 110 µL 50 µL
Volume to Hybridize 490 µL 240 µL 100 µL 40 µL

One-Color Microarray-Based Gene Expression Analysis (Low Input Quick Amp Labeling) Protocol 37
2 Procedures 
Step 3. Prepare the hybridization assembly

3 If any wells are unused:

a Make a 1× solution of the 2× Hi-RPM Hybridization Buffer.
b Add the volume of 1× Hybridization Buffer equal to the sample volume to
each unused well.
Make sure all wells contain sample or 1× Hybridization Buffer. Empty wells
can cause failure in hybridization.
4 Grip the slide on either end and slowly put the slide “active side” down,
parallel to the SureHyb gasket slide, so that the “Agilent”-labeled barcode is
facing down and the numeric barcode is facing up. Make sure that the
sandwich-pair is properly aligned.

Do not drop the array slide onto the gasket. Doing so increases the chances of
CAUTION
samples mixing between gasket wells.

5 Put the SureHyb chamber cover onto the sandwiched slides and slide the
clamp assembly onto both pieces.
6 Firmly hand-tighten the clamp onto the chamber.
7 Vertically rotate the assembled chamber to wet the gasket and assess the
mobility of the bubbles. If necessary, tap the assembly on a hard surface to
move stationary bubbles.

38 One-Color Microarray-Based Gene Expression Analysis (Low Input Quick Amp Labeling) Protocol
 Procedures 2
Step 4. Hybridize

Step 4. Hybridize
1 Load each assembled chamber into the oven rotator rack. Start from the
center of the rack (position 3 or 4 when counting from the left). Set your
hybridization rotator to rotate at 10 rpm when using 2× Hi-RPM
Hybridization Buffer.
2 Hybridize at 65°C for 17 hours.

If you are not loading all the available positions on the hybridization rotator rack, be
CAUTION
sure to balance the loaded hybridization chambers on the rack so that there are an
equal number of empty positions on each of the four rows on the hybridization rack.

The Gene Expression Wash Buffer 2 needs to be warmed overnight. Make sure that you
NOTE prepare the wash buffer the night before you plan to do the microarray wash. See “Step 2.
Prewarm Gene Expression Wash Buffer 2”.

One-Color Microarray-Based Gene Expression Analysis (Low Input Quick Amp Labeling) Protocol 39
2 Procedures 
Microarray Wash

Microarray Wash

Step 1. Add Triton X-102 to Gene Expression wash buffers

This step is optional but highly recommended.
The addition of 0.005% Triton X-102 (10%) to the Gene Expression wash
buffers reduces the possibility of array wash artifacts. Add Triton X-102 (10%)
to Gene Expression Wash Buffer 1 and Gene Expression Wash Buffer 2 when
the cubitainer of wash buffer is first opened.
Do this step to both Gene Expression Wash Buffer 1 and
Gene Expression Wash Buffer 2 before use.
1 Open the cardboard box with the cubitainer of wash buffer and carefully
remove the outer and inner caps from the cubitainer.
2 Use a pipette to add 2 mL of the provided Triton X-102 (10%) into the wash
buffer in the cubitainer.
3 Replace the original inner and outer caps and mix the buffer carefully but
thoroughly by inverting the container 5 to 6 times.
4 Carefully remove the outer and inner caps and install the spigot provided
with the wash buffer.
5 Prominently label the wash buffer box to indicate that Triton X-102 (10%)
has been added and indicate the date of addition.
Triton X-102 (10%) can be added to smaller volumes of wash buffer as long as
the final dilution of the 10% Triton X-102 is 0.005% in the Gene Expression
wash buffer solution.

40 One-Color Microarray-Based Gene Expression Analysis (Low Input Quick Amp Labeling) Protocol
 Procedures 2
Step 2. Prewarm Gene Expression Wash Buffer 2

Step 2. Prewarm Gene Expression Wash Buffer 2

Warm the Gene Expression Wash Buffer 2 to 37°C as follows:
1 Dispense 1000 mL of Gene Expression Wash Buffer 2 directly into a sterile
storage bottle. Repeat until you have enough prewarmed Wash Buffer 2
solution for your experiment.
2 Tightly cap the sterile storage bottle and put in a 37°C water bath the night
before washing arrays. Alternatively, remove the plastic cubitainer from the
box and put it in a 37°C water bath the night before washing the arrays.

Step 3. Prepare the equipment

Always use clean equipment when doing the hybridization and wash steps.
Designate and dedicate dishes to one-color experiments.

Solvent wash
Wash staining dishes, racks and stir bars with acetonitrile or isopropyl alcohol
to avoid wash artifacts on your slides and images.
• Use acetonitrile for equipment that was exposed to Stabilization and
Drying Solution.
• Use isopropyl alcohol for equipment that was not exposed to
Stabilization and Drying Solution.

Conduct solvent washes in a vented fume hood.

WA R N I N G

1 Add the slide rack and stir bar to the staining dish.
2 Transfer the staining dish with the slide rack and stir bar to a magnetic stir
plate.
3 Fill the staining dish with 100% acetonitrile or isopropyl alcohol.
4 Turn on the magnetic stir plate and adjust the speed to a setting of 4
(medium speed).
5 Wash for 5 minutes.
6 Discard the solvent as is appropriate for your site.

One-Color Microarray-Based Gene Expression Analysis (Low Input Quick Amp Labeling) Protocol 41
2 Procedures 
Step 3. Prepare the equipment

7 Repeat step 1 through step 6.

8 Air dry the staining dish in the vented fume hood.
9 Proceed to “Milli-Q water wash”.

Milli-Q water wash

Wash all dishes, racks, and stir bars with Milli-Q water.
1 Run copious amounts of Milli-Q water through the staining dish.
2 Empty out the water collected in the dish.
3 Repeat step 1 and step 2 at least 5 times, as it is necessary to remove any
traces of contaminating material.
4 Discard the Milli-Q water.

Some detergents may leave fluorescent residue on the dishes. Do not use any
CAUTION
detergent in the washing of the staining dishes. If detergent is used, all traces must
be removed by copiously rinsing with Milli-Q water.

42 One-Color Microarray-Based Gene Expression Analysis (Low Input Quick Amp Labeling) Protocol
 Procedures 2
Step 4. Wash the microarray slides

Step 4. Wash the microarray slides

The microarray wash procedure for the Agilent one-color platform must be done in
NOTE environments where ozone levels are 50 ppb or less. For Scanner C and Scanner B, if
ozone levels exceed 50 ppb in your laboratory, use the Agilent Ozone Barrier Slide Cover
(described in this topic). SureScan microarray scanner uses a slide holder with a built-in
ozone barrier.

When setting up the apparatus for the washes, be sure to do so near the water bath
NOTE containing the pre-warmed Wash 2 solutions.

Table 22 lists the wash conditions for the wash procedure.

Table 22 Wash conditions

Dish Wash Buffer Temperature Time

Disassembly 1 Gene Expression Wash Buffer 1 Room temperature
1st wash 2 Gene Expression Wash Buffer 1 Room temperature 1 minute
*
2nd wash 3 Gene Expression Wash Buffer 2 Elevated temperature 1 minute

* The elevated temperature of the second wash step is usually around 31°C due to cooling by the
room temperature dish and the rack of arrays.

1 Completely fill slide-staining dish #1 with Gene Expression Wash Buffer 1

at room temperature.
2 Put a slide rack into slide-staining dish #2. Add a magnetic stir bar. Fill
slide-staining dish #2 with enough Gene Expression Wash Buffer 1 at room
temperature to cover the slide rack. Put this dish on a magnetic stir plate.
3 Put the empty dish #3 on the stir plate and add a magnetic stir bar. Do not
add the prewarmed (37°C) Gene Expression Wash Buffer 2 until the first
wash step has begun.
4 Remove one hybridization chamber from incubator and record time. Record
whether bubbles formed during hybridization and if all bubbles are rotating
freely.

One-Color Microarray-Based Gene Expression Analysis (Low Input Quick Amp Labeling) Protocol 43
2 Procedures 
Step 4. Wash the microarray slides

5 Prepare the hybridization chamber disassembly.

a Put the hybridization chamber assembly on a flat surface and loosen the
thumbscrew, turning counterclockwise.
b Slide off the clamp assembly and remove the chamber cover.
c With gloved fingers, remove the array-gasket sandwich from the chamber
base by grabbing the slides from their ends. Keep the microarray slide
numeric barcode facing up as you quickly transfer the sandwich to
slide-staining dish #1.
d Without letting go of the slides, submerge the array-gasket sandwich into
slide-staining dish #1 containing Gene Expression Wash Buffer 1.
6 With the sandwich completely submerged in
Gene Expression Wash Buffer 1, pry the sandwich open from the barcode
end only:
a Slip one of the blunt ends of the forceps between the slides.
b Gently turn the forceps upwards or downwards to separate the slides.
c Let the gasket slide drop to the bottom of the staining dish.
d Grasp the top corner of the microarray slide, remove the slide, and then
put it into the slide rack in the slide-staining dish #2 that contains
Gene Expression Wash Buffer 1 at room temperature. Transfer the slide
quickly so avoid premature drying of the slides. Touch only the barcode
portion of the microarray slide or its edges!
More effort is needed to separate the 4-pack and 8-pack sandwiched slides
than the 1-pack and 2-pack sandwiched slides.
7 Repeat step 4 through step 6 for up to seven additional slides in the group.
For uniform washing, do up to a maximum of eight disassembly procedures
yielding eight microarray slides.
8 When all slides in the group are placed into the slide rack in slide-staining
dish #2, stir using setting 4 for 1 minute.
9 During this wash step, remove Gene Expression Wash Buffer 2 from the
37°C water bath and pour into the slide-staining dish #3.
10 Transfer slide rack to slide-staining dish #3 that contains
Gene Expression Wash Buffer 2 at elevated temperature. Stir using setting
4, or a moderate speed setting, for 1 minute.
11 Slowly remove the slide rack minimizing droplets on the slides. It should
take 5 to 10 seconds to remove the slide rack. If liquid remains on the
bottom edge of the slide, dab it on a cleaning tissue.

44 One-Color Microarray-Based Gene Expression Analysis (Low Input Quick Amp Labeling) Protocol
 Procedures 2
Step 4. Wash the microarray slides

12 Discard used Gene Expression Wash Buffer 1 and

Gene Expression Wash Buffer 2.
13 Repeat step 1 through step 12 for the next group of eight slides using fresh
Gene Expression Wash Buffer 1 and Gene Expression Wash Buffer 2
pre-warmed to 37°C.
14 Put the slides in a slide holder.
For SureScan microarray scanner
• Carefully put the end of the slide without the barcode label onto the slide
ledge.
• Gently lower the microarray slide into the slide holder. Make sure that
the active microarray surface (with “Agilent”-labeled barcode) faces up,
toward the slide cover.
• Close the plastic slide cover, pushing on the tab end until you hear it
click.
• For more detailed instruction, refer to the Agilent G4900DA SureScan
Microarray Scanner System User Guide.

Figure 3 Slide in slide holder for SureScan microarray scanner

For Agilent Scanner B or C only:

• In environments in which the ozone level exceeds 50 ppb, immediately
put the slides with active microarray surface (with “Agilent”-labeled
barcode) facing up in a slide holder. Make sure that the slide is not
caught up on any corner. Put an ozone-barrier slide cover on top of the
array as shown in Figure 4. Refer to the Agilent Ozone-Barrier Slide
Cover User Guide (p/n G2505-90550), included with the slide cover, for
more information.
As an alternative, use the Stabilization and Drying Solution. See
“Preventing Ozone-Related Problems” on page 79.

One-Color Microarray-Based Gene Expression Analysis (Low Input Quick Amp Labeling) Protocol 45
2 Procedures 
Step 4. Wash the microarray slides

Figure 4 Inserting the ozone-barrier slide cover (shown for Scanner B and Scanner C)

• In environments in which the ozone level is below 50 ppb, put the slides
with Agilent barcode facing up in a slide holder.
15 Scan slides immediately to minimize the impact of environmental oxidants
on signal intensities. If necessary, store slides in orange slide boxes in a
nitrogen purge box, in the dark.

46 One-Color Microarray-Based Gene Expression Analysis (Low Input Quick Amp Labeling) Protocol
 Procedures 2
Scanning and Feature Extraction

Scanning and Feature Extraction

Step 1. Scan the slides

Agilent provides support for Agilent microarrays scanned on select
non-Agilent scanners. Please see “Feature Extraction Compatibility Matrix for
Non Agilent scanners” for scanner compatibility and settings
([Link]
[Link]).
Agilent can guarantee the quality of data only if the data comes from Agilent
microarrays scanned on Agilent scanners.
A SureScan or Agilent C microarray scanner is required for SurePrint G3
formats.
To get scanner profiles from Agilent:
• For Scan Control 9.1.3 or later, go to
[Link]
• For Scan Control 8.x, go to
[Link]

Agilent SureScan Microarray Scanner

1 Put assembled slide holders into the scanner cassette.
2 Select the appropriate scanner protocol:
• AgilentG3_HiSen_GX_1color (for G3 format, high-sensitivity mode)
• AgilentG3_GX_1color (for G3 format, standard mode)
• AgilentHD_GX_1color (for HD format)
3 Verify that the Scanner status in the main window says Scanner Ready.
4 Click Start Scan.

One-Color Microarray-Based Gene Expression Analysis (Low Input Quick Amp Labeling) Protocol 47
2 Procedures 
Step 1. Scan the slides

Agilent C Scanner Settings

1 Put assembled slide holders with or without the ozone-barrier slide cover
into scanner carousel.
2 Select Start Slot m End Slot n where the letter m represents the Start slot
where the first slide is located and the letter n represents the End slot
where the last slide is located.
3 Select Profile AgilentG3_GX_1color (for SurePrint G3 formats) or Profile
AgilentHD_GX_1color (for SurePrint HD formats).
4 Verify scan settings for one-color scans. See Table 23.

Do not scan G3 microarrays with HD format settings. The resolution of the resulting
CAUTION
image will not be high enough for data analysis.

Table 23 C Scanner Scan Settings

For HD Microarray Formats For G3 Microarray Formats

Dye channel G (green) G (green)
Scan region Agilent HD (61 × 21.6 mm) Agilent HD (61 × 21.6 mm)
Scan resolution 5 µm 3 µm
Tiff file dynamic range 20 bit 20 bit
Green PMT gain 100% 100%

5 Verify that Output Path Browse is set for desired location.

6 Verify that the Scanner status in the main window says Scanner Ready.
7 Click Scan Slot m-n on the Scan Control main window where the letter m
represents the Start slot where the first slide is located and the letter n
represents the End slot where the last slide is located.

48 One-Color Microarray-Based Gene Expression Analysis (Low Input Quick Amp Labeling) Protocol
 Procedures 2
Step 1. Scan the slides

Agilent B Scanner Settings

Agilent Scan Control software v7.0.03 is recommended for 5 μm scans of
SurePrint HD formats. The Agilent B Scanner does not support G3
microarrays. For G3 microarrays, use the Agilent C Scanner or SureScan
microarray scanner.

1 Put slide into slide holder, with or without the ozone-barrier slide cover,
with Agilent barcode facing up.
2 Put assembled slide holders into scanner carousel.
3 Verify scan settings for one-color scans. See Table 24.
For version 7.X, to change any settings, click Settings > Modify Default
Settings. A window pops up from which you can change the settings.

Table 24 B Scanner Scan Settings

For All Formats

Scan region Scan Area (61 × 21.6 mm)
Scan resolution (µm) 5
5µm scanning mode Single Pass
eXtended Dynamic range (selected)
Dye channel Green
Green PMT XDR Hi 100%
XDR Lo 10%

4 Select settings for the automatic file naming.

• Prefix1 is set to Instrument Serial Number.
• Prefix2 is set to Array Barcode.
5 Verify that the Scanner status in the main window says Scanner Ready.
6 Click Scan Slot m-n on the Scan Control main window where the letter m
represents the Start slot where the first slide is located and the letter n
represents the End slot where the last slide is located.

One-Color Microarray-Based Gene Expression Analysis (Low Input Quick Amp Labeling) Protocol 49
2 Procedures 
Step 2. Extract data using Agilent Feature Extraction Software

Step 2. Extract data using Agilent Feature Extraction Software

Feature Extraction is the process by which information from probe features is
extracted from microarray scan data, allowing researchers to measure gene
expression in their experiments. To get the most recent Feature Extraction
software for gene expression, go to the Agilent web site at
[Link]/chem/fe.
After generating the microarray scan images, extract .tif images using the
Feature Extraction software.

1 Open the Agilent Feature Extraction (FE) program.

To get the most recent Feature Extraction protocols for gene expression, go
to [Link]/chem/feprotocols.
2 Add the images (.tif) to be extracted to the FE Project.
a Click Add New Extraction Set(s) icon on the toolbar or right-click the
Project Explorer and select Add Extraction...
b Browse to the location of the .tif files, select the .tif file(s) and click
Open. To select multiple files, use the Shift or Ctrl key when selecting.
The FE program automatically assigns a default grid template and protocol
for each extraction set, if the following conditions are met:
• For auto assignment of the grid template, the image must be generated
from a Agilent scanner and have an Agilent barcode.
• For auto assignment of the One-Color Gene Expression FE protocol, the
default Gene Expression protocol must be specified in the FE Grid
Template properties.
To access the FE Grid Template properties, double-click on the grid
template in the Grid Template Browser.
3 Set FE Project Properties.
a Select the Project Properties tab.
b In the General section, enter your name in the Operator text box.
c In the Input section, verify that at least the following default settings as
shown in Figure 5 are selected.
For outputs that can be imported into Rosetta Resolver, select MAGE and
JPEG.

50 One-Color Microarray-Based Gene Expression Analysis (Low Input Quick Amp Labeling) Protocol
 Procedures 2
Step 2. Extract data using Agilent Feature Extraction Software

Figure 5 Default settings in FE 10.7.

4 Check the Extraction Set Configuration.

a Select the Extraction Set Configuration tab.
b Verify that the correct grid template is assigned to each extraction set in
the Grid Name column. To assign a different grid template to an
extraction set, select one from the pull down menu.
If a grid template is not available to select from the pull down menu, you
must add it to the Grid Template Browser. To add, right-click inside the
Grid Template Browser, select Add. Browse for the design file (.xml) and
click Open to load grid template into the FE database.
To update to the latest grid templates via Online Update, right-click Grid
Template Browser and select Online Update. You can also download the
latest grid templates from Agilent web site at
[Link] After downloading, you must add the
grid templates to the Grid Template Browser.
After a new grid template is added to the Grid Template Browser,
remember to specify the default protocol for the new grid template if you
want the Feature Extraction program to automatically assign a FE
protocol to an extraction set.
c Verify that the correct protocol is assigned to each extraction set in the
Protocol Name column. To assign a different protocol to an extraction

One-Color Microarray-Based Gene Expression Analysis (Low Input Quick Amp Labeling) Protocol 51
2 Procedures 
Step 2. Extract data using Agilent Feature Extraction Software

set, select from the pull down menu. The appropriate protocol begins
with “GE1” for one-color analysis.
The protocols automatically distinguish the formats for processing the
data.
If a protocol is not available to select from the pull down menu, you must
import it to the FE Protocol Browser. To import, right-click FE Protocol
Browser, select Import. Browse for the FE protocol (.xml) and click
Open to load the protocol into the FE database. Visit the Agilent web site
at [Link]/chem/feprotocols to download the latest protocols.

These FE Protocols were optimized using data from Agilent catalog arrays, which have
NOTE many replicated probes and validated Negative Control probes. If custom arrays without
enough replicated probes are used, or arrays with custom probes designated as Negative
Control probes are used, the default FE Protocols may not be optimal.

When the Agilent XDR scanned images are added to Feature Extraction software version
NOTE 9.1 or later, the High and Low images are automatically combined for data extraction.

20-bit single images from the C Scanner are equivalent to 16-bit XDR images from the B
NOTE Scanner.

5 Save the FE Project (.fep) by selecting File > Save As and browse for
desired location.
6 Verify that the icons for the image files in the FE Project Window no longer
have a red X through them. A red X through the icon indicates that an
extraction protocol was not selected. If needed, reselect the extraction
protocol for that image file.
7 Select Project > Start Extracting.
8 After the extraction is completed successfully, view the QC report for each
extraction set by double-clicking the QC Report link in the Summary
Report tab. Determine whether the grid has been properly placed by
inspecting Spot Finding at the Four Corners of the Array. See Figure 7 and
Figure 8.

52 One-Color Microarray-Based Gene Expression Analysis (Low Input Quick Amp Labeling) Protocol
 Procedures 2
Step 2. Extract data using Agilent Feature Extraction Software

If a QC Metric Set has been assigned to the FE Project, you can view the results
of the metric evaluation in three ways:
• Project Run Summary - includes a summary sentence.
• QC Report - includes both a summary on the header and a table of metric
values.
• QC Chart - includes a view of the values of each metric compared across all
extractions in FE Project.
Refer to the application note Enhanced Quality Assessment Using Agilent
Feature Extraction QC Metric Sets, Thresholds, and Charting Tools (p/n
5989-5952EN) for more details on quality assessment and troubleshooting
with the Feature Extraction QC Report. This technical note can be
downloaded from the Agilent web site at [Link]. Search for the part
number 5989-5952EN.

Automatic Download from eArray

Feature Extraction version 10.7 or higher can automatically download Grid
Templates, protocols and QC metrics (QCM or QCMT). To set this up, in the
eArray Login Setting dialog box, under Advanced Options, click Use eArray
server during extraction. See Figure 6.

Figure 6 eArray Login Setting

One-Color Microarray-Based Gene Expression Analysis (Low Input Quick Amp Labeling) Protocol 53
2 Procedures 
Step 2. Extract data using Agilent Feature Extraction Software

Page 1 of 3

QC Report - Agilent Technologies : 1 Color Gene Expression

Date Tuesday, January 10, 2012 - 17:31 Grid 028004_D_F_20111107

Image US23502418_252800413846_S01 [2_2] BG Method No Background

Protocol GE1_107_Sep09 (Read Only) Background Detrend On(FeatNCRange, LoPass)

User Name annlucas Multiplicative Detrend True

FE Version [Link] Additive Error 3(Green)

Sample(red/green) Saturation Value 777466 (g)

Spot Finding of the Four Corners of the Array Net Signal Statistics
Agilent SpikeIns:
Green

# Saturated Features 0
99% of Sig. Distrib. 291929
50% of Sig. Distrib. 1246
1% of Sig. Distrib. 23

Non-Control probes:
Grid Normal Green
Local
Feature
Background
# Saturated Features 0
Green Green
99% of Sig. Distrib. 34968
50% of Sig. Distrib. 177
Non Uniform 4 3
1% of Sig. Distrib. 20
Population 371 3686
Histogram of Signals Plot
Spatial Distribution of All Outliers on the Array
384 rows x 164 columns

# Features (NonCtrl) with BGSubSignal < 0: 4252 (Green)

# FeatureNonUnif (Green) = 4(0.01%)

# GeneNonUnif (Green) = 0 (0.000 %)

•BG NonUniform •BG Population

•Green FeaturePopulation •Green Feature NonUniform

Negative Control Stats

Green

Average Net Signals 21.42

Figure 7 Example of the first page of a QC Report for 8×60K microarray, generated by
Feature Extraction Software

54 One-Color Microarray-Based Gene Expression Analysis (Low Input Quick Amp Labeling) Protocol
 Procedures 2
Step 2. Extract data using Agilent Feature Extraction Software

QC Report - Agilent Technologies : 1 Color Gene Expression

Date Tuesday, November 24, 2009 - 15:34 Grid 014850_D_F_20090416

Image US23502418_251485048994_S01 [1_4] BG Method No Background

Protocol GE1_107_Sep09 (Read Only) Background Detrend On(FeatNCRange, LoPass)

User Name Administrator Multiplicative Detrend True

FE Version [Link] Additive Error 1(Green)

Sample(red/green) Saturation Value 777631 (g)

Spot Finding of the Four Corners of the Array Net Signal Statistics
Agilent SpikeIns:
Green

# Saturated Features 0
99% of Sig. Distrib. 258889
50% of Sig. Distrib. 1167
1% of Sig. Distrib. 26

Non-Control probes:
Grid Normal Green
Local
Feature
Background
# Saturated Features 0
Green Green
99% of Sig. Distrib. 29234
50% of Sig. Distrib. 185
Non Uniform 0 10
1% of Sig. Distrib. 21
Population 91 1172
Histogram of Signals Plot
Spatial Distribution of All Outliers on the Array
532 rows x 85 columns

# Features (NonCtrl) with BGSubSignal < 0: 1221 (Green)

# FeatureNonUnif (Green) = 0(0.00%)

# GeneNonUnif (Green) = 0 (0.000 %)

•BG NonUniform •BG Population

•Green FeaturePopulation •Green Feature NonUniform

Figure 8 Example of the first page of a QC Report for 4×44K microarray, generated by
Feature Extraction Software

One-Color Microarray-Based Gene Expression Analysis (Low Input Quick Amp Labeling) Protocol 55
 This page intentionally left blank.

56 One-Color Microarray-Based Gene Expression Analysis (Low Input Quick Amp Labeling) Protocol
 One-Color Microarray-Based Gene Expression Analysis
Protocol

3
Supplemental Procedures
Absolutely RNA Nanoprep Purification 58
Thermocycler Protocol 61
Quick Amp Labeling Kit Sample Preparation 64
Quality Assessment of Template RNA and Labeled cRNA 73
Preventing Ozone-Related Problems 79
Normalizing Agilent One-Color Microarray Data 84

The procedures in this chapter are optional but recommended.

57
3 Supplemental Procedures 
Absolutely RNA Nanoprep Purification

Absolutely RNA Nanoprep Purification

As an alternative to the RNeasy Mini Kit, the Absolutely RNA Nanoprep Kit
can be used to purify the amplified cRNA after “Step 2. Prepare labeling
reaction” on page 26. Use the Absolutely RNA Nanoprep Kit when it is
required or to avoid the need to concentrate purified samples. The Absolutely
RNA Nanoprep Kit uses an elution volume of 20 μL.

Step 1. Prepare the reagents

1 Prepare 80% sulfolane:
a Incubate the 100% sulfolane in a 37°C water bath until liquefied.
100% sulfolane is a solid at room temperature. 80% sulfolane solution is a
liquid at room temperature and can be stored at room temperature for at
least a month.
b Add 1 mL of DNase/RNase-free distilled water to 4 mL of 100% sulfolane
to make 5 mL of 80% sulfolane.
5 mL of 80% sulfolane is enough to process 50 RNA preparations (from up
to 0.1 mL lysate each).
2 Prepare 1× high-salt wash buffer:
a Add 16 mL of 100% ethanol to the bottle of 1.67× High Salt Wash Buffer.
b On the 1.67× High Salt Wash Buffer container, mark the check box for 1×
(Ethanol Added).
c Tighten the cap on the container of 1.67× High Salt Wash Buffer and
store at room temperature.
3 Prepare the 1× low-salt wash buffer:
a Add 68 mL of 100% ethanol to the bottle of 5× Low Salt Wash Buffer.
b On the 5× Low Salt Wash Buffer container, mark the check box for 1×
(Ethanol Added).
c Tighten the cap on the container of 5× Low Salt Wash Buffer and store at
room temperature.

58 One-Color Microarray-Based Gene Expression Analysis (Low Input Quick Amp Labeling) Protocol
 Supplemental Procedures 3
Step 2. Purify the labeled/amplified RNA

Step 2. Purify the labeled/amplified RNA

1 Add 100 μL of the Lysis Buffer to each reaction tube for a total volume of
116 μL.
2 Mix on a vortex mixer, or pipette repeatedly until homogenized.
3 Add an equal volume (116 μL) of 80% sulfolane (room temperature) to the
cell lysate. Mix thoroughly on a vortex mixer for 5 seconds.
You must use equal volumes of 80% sulfolane and cell lysate. Mix on a
vortex mixer until the lysate and sulfolane are thoroughly mixed.
4 Put an RNA-binding nano-spin cup into a 2-ml collection tube.
5 Transfer the 80% sulfolane and cell lysate mixture to the RNA-binding
nano-spin cup and snap the RNA Binding Nano Spin Cup Cap onto the top
of the spin cup.
6 Spin the sample in a microcentrifuge at 12,000 rpm for 60 seconds.
7 Remove and keep the RNA-binding nano-spin cup. Discard the filtrate. Put
the RNA-binding nano-spin cup back into the same 2-ml collection tube.
Up to this point, the RNA has been protected from RNases by the presence
of guanidine thiocyanate.
8 Add 300 μL of 1× High-Salt Wash Buffer to the RNA-binding nano-spin cup.
Cap the RNA-binding nano-spin cup, and spin the sample in a
microcentrifuge at 12,000 rpm for 60 seconds.

The High-Salt Wash Buffer contains the irritant guanidine thiocyanate.

CAUTION

9 Remove and keep the spin cup. Discard the filtrate. Put the spin cup back
into the same 2-mL collection tube.
10 Add 300 μL of 1× Low-Salt Wash Buffer to the RNA-binding nano-spin cup.
Cap the spin cup, and spin the sample in a microcentrifuge at 12,000 rpm
for 60 seconds.
11 Repeat step 9 and step 10 for a second low-salt wash.
12 Remove and keep the spin cup. Discard the filtrate. Put the RNA-binding
nano-spin cup back into the same 2-ml collection tube.
13 Add 300 μL of 1× Low-Salt Wash Buffer to the RNA-binding nano-spin cup.
Cap the RNA-binding nano-spin cup, and spin the sample in a
microcentrifuge at 12,000 rpm for 3 minutes to dry the fiber matrix.

One-Color Microarray-Based Gene Expression Analysis (Low Input Quick Amp Labeling) Protocol 59
3 Supplemental Procedures 
Step 2. Purify the labeled/amplified RNA

14 Transfer the spin cup to a fresh 2-ml collection tube.

15 Add 20 μL of Elution Buffer directly onto the fiber matrix inside the
RNA-binding nano-spin cup. Cap the RNA-binding nano-spin cup and
incubate the sample at room temperature for 2 minutes.

The Elution Buffer must be added directly onto the fiber matrix so that the buffer can
NOTE permeate the entire fiber matrix.

To increase the RNA yield, warm the Elution Buffer to 60°C.

16 Spin the sample in a microcentrifuge at 12,000 rpm for 5 minutes.
17 If needed, repeat the elution step (step 15 and step 16) to increase the yield
of total RNA.
18 Transfer the eluate in the collection tube to a capped microcentrifuge tube
to store the RNA.
The RNA can be stored at -20°C for up to one month, or at -80°C for
long-term storage.

60 One-Color Microarray-Based Gene Expression Analysis (Low Input Quick Amp Labeling) Protocol
 Supplemental Procedures 3
Thermocycler Protocol

Thermocycler Protocol
The procedure in this section is an optional thermocycler protocol for the Low
Input Quick Amp Labeling Kit, One-Color.
Use a thermocycler to label reactions if you have a limited number of water
baths. The use of a thermocycler can slightly lower the yield of cRNA when
compared to the use of water baths.

Step 1. Program the thermocycler

• Store the following programs into your thermocycler:
• Program 1: 65°C for 10 minutes, 4°C hold
• Program 2: 40°C for 2 hours, 70°C for 15 minutes, 4°C hold
• Program 3: 40°C for 2 hours, 4°C hold
Five minutes at 4°C is enough. Hold at that temperature if the reagents for the
next step are not ready.

Use a heated lid for optimal results.

NOTE

One-Color Microarray-Based Gene Expression Analysis (Low Input Quick Amp Labeling) Protocol 61
3 Supplemental Procedures 
Step 2. Synthesize cDNA from Total RNA

Step 2. Synthesize cDNA from Total RNA

(Time required: ~3 hours)
1 Add 25 to 200 ng of total RNA to a 0.2 mL PCR tube or the well of a 96-well
PCR plate in a volume of 1.5 μL. For optimal performance, use at least 25 ng
of input total RNA.
2 Add 2 μL of the diluted Spike Mix. Please refer to Table 13 on page 23 for
detailed instructions on the preparation and use of Spike-in kits.
3 Prepare the T7 Primer (green cap) Master Mix as described in Table 14 on
page 27.
4 Add 1.8 μL of T7 Promoter Primer Mix to the tube that contains 3.5 μL of
total RNA and diluted RNA spike-in controls. Each tube now contains a
total volume of 5.3 μL.
5 Put the tubes in the thermocycler and run Program 1 to denature the
template and anneal the primer.
6 Keep the reaction tubes in the thermocycler at 4°C, or move to bench top
rack on ice.
7 Immediately prior to use, gently mix the components in Table 15 on page 28
in the order listed by pipetting, and keep at room temperature.

Prewarm the 5× First Strand Buffer (green cap) by incubating the vial in an 80°C water bath
NOTE for 3 to 4 minutes to ensure adequate resuspension of the buffer components. For optimal
resuspension, mix briefly on a vortex mixer and spin the tube briefly in a microcentrifuge to
drive the contents off the tube walls. Keep at room temperature until use.

Keep the Affinity Script RNase Block Mix (violet cap) on ice. Do not add the Affinity Script RNase
NOTE Block Mix (violet cap) until just before you start the reactions.

8 To each sample tube, add 4.7 μL of cDNA Master Mix for a total volume of
10 μL. Pipette up and down to mix.
9 Put reaction tubes in thermocycler and run Program 2 to synthesize
double-stranded cDNA.

Incubation at 70°C inactivates the AffinityScript enzyme.

NOTE

62 One-Color Microarray-Based Gene Expression Analysis (Low Input Quick Amp Labeling) Protocol
 Supplemental Procedures 3
Step 3. Synthesize Fluorescent cRNA Synthesis in vitro

Step 3. Synthesize Fluorescent cRNA Synthesis in vitro

(Time required: ~2.5 hours)
1 Immediately before use, make Master Mix for each cyanine dye:
a Add the first four components listed in Table 16 on page 29 in the order
shown to 1.5-mL nuclease-free microfuge tubes at room temperature.
b Mix thoroughly on a vortex mixer.
c Add the T7 RNA Polymerase Blend (red cap) and Cyanine 3-CTP.
d Mix gently, but completely, by pipetting up and down without introducing
bubbles.

Do not add the T7 RNA Polymerase Blend (red cap) to Transcription Master Mix until just
NOTE before you do the reaction.

2 Keep the reaction tubes from step 9 above in the thermocycler at 4°C, or
move to bench top rack on ice.
3 To each sample tube, add 6 μL of Transcription Master Mix. Gently mix by
pipetting up and down. The final volume of the reaction is now 16 μL.
4 Return the reaction tubes to the thermocycler and run Program 3 (“Step 1.
Program the thermocycler” on page 61) to synthesize labeled cRNA.
5 Purify the labeled cRNA as described on “Step 3. Purify the
labeled/amplified RNA” on page 30.

One-Color Microarray-Based Gene Expression Analysis (Low Input Quick Amp Labeling) Protocol 63
3 Supplemental Procedures 
Quick Amp Labeling Kit Sample Preparation

Quick Amp Labeling Kit Sample Preparation

The Low Input Quick Amp Labeling Kit replaces the Quick Amp Labeling Kit,
One-Color. If you have studies that are ongoing, continue to use the Quick Amp
Labeling preparation steps described in this section. For new studies, use the
Low Input Quick Amp Labeling Kit as described in Chapter 2, “Procedures”.
The Quick Amp Labeling Kit, One-Color generates fluorescent cRNA
(complimentary RNA) with a sample input RNA range between 200 ng and
1 μg of total RNA or a minimum of 10 ng of poly A+ RNA for one-color
processing. The method uses T7 RNA Polymerase Blend (red cap), which
simultaneously amplifies target material and incorporates Cyanine 3-CTP.
There is routinely at least a 100-fold RNA amplification with use of this kit.

For optimal performance, use high quality, intact template total or poly A+ RNA. Please
NOTE refer to “Quality Assessment of Template RNA and Labeled cRNA” on page 73 for general
guidance and procedural recommendations on quality assessment of template RNA.

64 One-Color Microarray-Based Gene Expression Analysis (Low Input Quick Amp Labeling) Protocol
 Supplemental Procedures 3
Quick Amp Labeling Kit Sample Preparation

Figure 9 Schematic of amplified cRNA procedure. Generation of cRNA for a two-color

microarray experiment is shown. When you generate targets for a one-color
microarray experiment, only the Cy3-labeled “A” sample is produced and
hybridized.

One-Color Microarray-Based Gene Expression Analysis (Low Input Quick Amp Labeling) Protocol 65
3 Supplemental Procedures 
Step 1. Prepare Spike Mix

Step 1. Prepare Spike Mix

Refer to the protocol for the RNA Spike-In Kit, One-Color for in-depth
instructions and troubleshooting advice on how to use the spike mix. This
protocol is available with the RNA Spike-In Kit, One-Color and can also be
downloaded from the Agilent web site at
[Link]/chem/dnamanuals-protocols.

1 Equilibrate water baths to 37°C, 65°C, 40°C, and 80°C.

2 Mix the Spike Mix solution vigorously on a vortex mixer.
3 Heat at 37°C for 5 minutes, and mix on a vortex mixer once more.
4 Briefly spin in a centrifuge to drive contents to the bottom of the tube prior
to opening. Settlement of the solution on the sides or lid of the tubes may
occur during shipment and storage.
Table 25 provides the dilutions of Spike Mix for a range of total RNA input
amounts. These are diluted such that 1 μL of Spike Mix is added for every
100 ng of total RNA in the labeling reaction up to 500 ng, and 0.5 μL added for
every 100 ng total RNA for input amounts greater than 500 ng. If you start
with the minimum 10 ng mRNA as the input mass, follow the dilution scheme
as described for the input mass of 1000 ng total RNA.

66 One-Color Microarray-Based Gene Expression Analysis (Low Input Quick Amp Labeling) Protocol
 Supplemental Procedures 3
Step 1. Prepare Spike Mix

Table 25 Dilutions of Agilent One-Color Spike Mix for Cyanine 3-labeling

Starting Amount of RNA Serial Dilution Spike Mix Volume to be used

in each labeling reaction (µL)

Total RNA PolyA Maximum Volume First Second Third

(ng) RNA (ng) of RNA (µL)

200 8.3 1:20 1:25 1:10 2

300 7.3 1:20 1:25 1:10 3

400 6.3 1:20 1:25 1:10 4

500 5.3 1:20 1:25 1:10 5

600 7.3 1:20 1:25 1:5 3

700 6.8 1:20 1:25 1:5 3.5

800 6.3 1:20 1:25 1:5 4

900 5.8 1:20 1:25 1:5 4.5

1000 10 5.3 1:20 1:25 1:5 5

Use RNase-free microfuge tubes and tips. Avoid pipetting volumes less than 2 µL to
NOTE ensure accuracy.

For example, to prepare the Spike Mix dilution appropriate for 200 ng of total
RNA starting sample:
1 Make the First Dilution:
a Mix the thawed Spike Mix vigorously on a vortex mixer.
b Heat at 37°C in a circulating water bath for 5 minutes.
c Mix the Spike Mix tube vigorously again on a vortex mixer.
a Label a new sterile 1.5 mL microcentrifuge tube “Spike Mix First
Dilution.”
b Into the First Dilution tube, put 2 μL of the concentrated Spike Mix.
c Add 38 μL of the Dilution Buffer (1:20).
d Mix thoroughly on a vortex mixer and spin down quickly in a centrifuge
to collect all of the liquid at the bottom of the tube. This tube contains
the First Dilution.

One-Color Microarray-Based Gene Expression Analysis (Low Input Quick Amp Labeling) Protocol 67
3 Supplemental Procedures 
Step 1. Prepare Spike Mix

2 Make the Second Dilution:

a Label a new sterile 1.5 mL microcentrifuge tube “Spike Mix Second
Dilution.”
b Into the Second Dilution tube, put 2 μL from the First Dilution tube.
c Add 48 μL of the Dilution Buffer (1:25).
d Mix thoroughly on a vortex mixer and spin down quickly in a centrifuge
to collect all of the liquid at the bottom of the tube. This tube contains
the Second Dilution.
3 Make the Third Dilution:
a Label a new sterile 1.5 mL microcentrifuge tube “Spike Mix Third
Dilution.”
b Into the Third Dilution tube, put 4 μL from the Second Dilution tube.
c Add 36 μL of the Dilution Buffer (1:10).
d Mix thoroughly on a vortex mixer and spin down quickly in a centrifuge
to collect all of the liquid at the bottom of the tube. This tube contains
the Third Dilution.
4 Add 2 μL of Third Dilution to 200 ng of sample total RNA and continue with
cyanine 3 labeling using the Quick Amp Labeling Kit as described in “Step 2.
Prepare labeling reaction” on page 69.

Storage of Spike Mix dilutions

Store the RNA Spike-In Kit, One-Color at –70°C to –80°C in a non-defrosting
freezer for up to 1 year from the date of receipt.
The first dilution of the Spike Mix positive controls can be stored up to 2
months in a non-defrosting freezer at –70°C to –80°C and freeze/thawed up to
eight times.
After use, discard the second and third dilution tubes.

68 One-Color Microarray-Based Gene Expression Analysis (Low Input Quick Amp Labeling) Protocol
 Supplemental Procedures 3
Step 2. Prepare labeling reaction

Step 2. Prepare labeling reaction

1 Add 200 to 1000 ng of total RNA to a 1.5-mL microcentrifuge tube in an
appropriate volume (see Table 26). Dilute samples so that at least 2 μL of
sample is pipetted into the tube.
2 Add 1.2 μL of T7 Promoter Primer (green cap). See Table 26.
3 Add the appropriate volume of diluted Spike Mix as indicated in Table 26.

Table 26 Template and T7 Promoter Primer Mix

Total RNA  PolyA Max RNA Third Dilution of Spike T7 Promoter Total
input (ng) RNA (ng) volume (µL) Mix volume (µL) primer (µL) volume
(µL)

200 8.3 2 1.2 11.5

300 7.3 3 1.2 11.5

400 6.3 4 1.2 11.5

500 5.3 5 1.2 11.5

600 7.3 3 1.2 11.5

700 6.8 3.5 1.2 11.5

800 6.3 4 1.2 11.5

900 5.8 4.5 1.2 11.5

1000 10 5.3 5 1.2 11.5

4 Use nuclease-free water to bring the total reaction volume to 11.5 μL.
5 Denature the primer and the template by incubating the reaction at 65°C in
a circulating water bath for 10 minutes.
6 Put the reactions on ice and incubate for 5 minutes.
7 Immediately prior to use, gently mix the components listed in Table 27 for
the cDNA Master Mix by adding in the order indicated, and keep at room
temperature.
8 Prewarm the 5× First Strand Buffer (green cap) at 80°C for 3 to 4 minutes
to ensure adequate resuspensions of the buffer components. For optimal
resuspension, briefly mix on a vortex mixer and spin the tube in a

One-Color Microarray-Based Gene Expression Analysis (Low Input Quick Amp Labeling) Protocol 69
3 Supplemental Procedures 
Step 2. Prepare labeling reaction

microcentrifuge to drive down the contents from the tube walls. Keep at
room temperature until needed.
MMLV-RT (violet cap) and RNase Inhibitor (violet cap) are enzymes, which
need to be kept on ice and are to be added to the cDNA Master Mix just
before starting the reactions.
Be sure to use the 10 mM dNTP mix tube from the kit.

Table 27 cDNA Master Mix

Component Volume (µL) per Volume (µL) per 4.5

reaction reactions

5× First Strand Buffer (green cap) 4 18

0.1 M DTT (white cap) 2 9

10 mM dNTP Mix (green cap) 1 4.5

MMLV-RT (violet cap) 1 4.5

RNase Inhibitor (violet cap) 0.5 2.3

Total Volume 8.5 38.3

9 Briefly spin each sample tube in a microcentrifuge to drive down the

contents from the tube walls and the lid.
10 Add 8.5 μL of cDNA Master Mix to each sample tube and mix by pipetting
up and down.
11 Incubate samples at 40°C in a circulating water bath for 2 hours.
12 Move samples to a 65°C circulating water bath and incubate for 15 minutes.
13 Move samples to ice. Incubate for 5 minutes.
14 Spin samples briefly in a microcentrifuge to drive down tube contents from
the tube walls and lid.
15 Immediately prior to use, gently mix the components listed in Table 28 in
the order indicated for the Transcription Master Mix by pipetting at room
temperature.
16 Prewarm the 50% PEG (clear cap) solution at 40°C for 1 minute. For
optimal resuspension, briefly mix on a vortex mixer and spin the tube in a
microcentrifuge to drive down the contents from the tube walls. Careful
pipetting is required to ensure accurate volume. Keep at room temperature
until needed.

70 One-Color Microarray-Based Gene Expression Analysis (Low Input Quick Amp Labeling) Protocol
 Supplemental Procedures 3
Step 2. Prepare labeling reaction

RNase Inhibitor (violet cap), inorganic pyrophosphatase, and T7 RNA

polymerase are enzymes, which need to be kept on ice and should be added
to the Transcription Master Mix just before starting the reactions.

Table 28 Transcription Master Mix

Component Volume (µL) per Volume (µL) per 4.5

reaction reactions

Nuclease-free water 15.3 68.9

4× Transcription Buffer (clear cap) 20 90

0.1 M DTT (white cap) 6 27

NTP Mix (blue cap) 8 36

PEG (clear cap) 6.4 28.8

RNase Inhibitor (violet cap) 0.5 2.3

Inorganic Pyrophosphatase (red cap) 0.6 2.7

T7 RNA Polymerase Blend (red cap) 0.8 3.6

Cyanine 3-CTP 2.4 10.8

Total Volume 60 270

17 Add 60 μL of Transcription Master Mix to each sample tube. Gently mix by

pipetting.
18 Incubate samples in a circulating water bath at 40°C for 2 hours.

One-Color Microarray-Based Gene Expression Analysis (Low Input Quick Amp Labeling) Protocol 71
3 Supplemental Procedures 
Step 3. Purify the labeled/amplified RNA

Step 3. Purify the labeled/amplified RNA

Use the RNeasy Mini Kit to purify the amplified cRNA samples.
If sample concentration causes difficulty, you can use the Absolutely RNA
Nanoprep Kit as an alternative. See “Absolutely RNA Nanoprep
Purification” on page 58.

Ensure that ethanol was added to the RPE buffer as specified in the Qiagen manual before
NOTE proceeding.

1 Add 20 μL of nuclease-free water to your cRNA sample, for a total volume of

100 μL.
2 Go to “Step 3. Purify the labeled/amplified RNA” on page 30. Start from
step 2 to complete this task.

Step 4. Quantify the cRNA

Use the NanoDrop ND-1000 UV-VIS Spectrophotometer version 3.2.1 to
quantify the cRNA.

1 Go to “Step 4. Quantify the cRNA” on page 32 and complete step 2 through

step 10
2 Examine the yield and specific activity results.

If the yield is <1.65 µg and the specific activity is <9.0 pmol Cy3 per µg cRNA do not
CAUTION proceed to the hybridization step. Repeat cRNA preparation.

Please refer to “Quality Assessment of Template RNA and Labeled cRNA” on page 73 for
NOTE general guidance and procedural recommendations on quality assessment of labeled
cRNA.

3 Continue to the hybridization step at “Hybridization” on page 34.

72 One-Color Microarray-Based Gene Expression Analysis (Low Input Quick Amp Labeling) Protocol
 Supplemental Procedures 3
Quality Assessment of Template RNA and Labeled cRNA

Quality Assessment of Template RNA and Labeled cRNA

This section gives a general guideline for template RNA and labeled cRNA
quality assessment before proceeding with amplification or hybridization.
Although optional, this step is highly recommended.
Make sure you determine the integrity and purity of the input template RNA,
as well as labeled cRNA, before you label/amplify and hybridize respectively.
Use the NanoDrop UV-VIS Spectrophotometer and the Agilent 2100
bioanalyzer. The RNA 6000 Nano LabChip kit can be used to analyze total
RNA, mRNA, or cRNA with the appropriate assay at the assay specified
concentration. For low concentration samples consider using the RNA 6000
Pico LabChip kit.
For the assessment of total RNA quality, the Agilent 2100 Expert Software
automatically provides a RNA Integrity Number (RIN). RIN provides a
quantitative value for RNA integrity that facilitates the standardization of
quality interpretation. Users should define a minimum threshold RIN number
based on correlative data in order to eliminate experimental bias due to poor
RNA quality. Analysis of single stranded RNA, e.g. mRNA and cRNA, provides
information on size distribution and concentration. It allows relative
quantification of fragments within a size range.

One-Color Microarray-Based Gene Expression Analysis (Low Input Quick Amp Labeling) Protocol 73
3 Supplemental Procedures 
Step 1. Prepare for quality assessment

Step 1. Prepare for quality assessment

• Refer to Table 29 and Table 30 to make sure that you have the appropriate
analyzer, kits, and compatible assays.

Table 29 Analyzer and Kits

Description Vendor and part number

2100 Bioanalyzer Agilent p/n G2938C or G2939A

RNA 6000 Nano LabChip Kit Agilent p/n 5067-1511

RNA 6000 Pico LabChip Kit Agilent p/n 5067-1513

Spectrophotometer NanoDrop p/n ND-1000 or equivalent

Table 30 Compatible Assays

Description Compatible Assay

RNA 6000 Nano LabChip Kit Eukaryote Total RNA Nano Assay Qualitative
range 5 to 500 ng/µL

RNA 6000 Nano LabChip Kit mRNA Nano Assay*

Qualitative range 25 to 250 ng/µL

RNA 6000 Pico LabChip Kit Eukaryote Total RNA Pico Assay Qualitative
range 50 to 5000 pg/µL in water

RNA 6000 Pico LabChip Kit mRNA Pico Assay*

Qualitative range 250 to 5000 pg/µL in water

* The mRNA assays are suitable for analysis of cRNA as well.

74 One-Color Microarray-Based Gene Expression Analysis (Low Input Quick Amp Labeling) Protocol
 Supplemental Procedures 3
Step 2. Assess the quality using the Agilent 2100 Bioanalyzer

Step 2. Assess the quality using the Agilent 2100 Bioanalyzer

1 Choose the kit and assay according to your needs. Typically the RNA
Nano 6000 kit and assay will be appropriate.
2 Ensure the 2100 bioanalyzer electrodes have been cleaned as instructed in
the reagent kit guide.
3 Start the Agilent 2100 Expert program (version B.02.06 or higher), turn on
the 2100 bioanalyzer and check communication.
4 Prepare the chip, samples and ladder as instructed in the reagent kit guide.
5 Load the prepared chip into the 2100 bioanalyzer and start the run within
five minutes after preparation.
6 Within the instrument context, choose the appropriate assay from the drop
down list.
7 Start the run. Enter sample names and comments in the Data and Assay
context.
8 Verify the results.

Template RNA results (total RNA)

The resulting electropherogram should have at least two distinct peaks
representing the 18S and 28S ribosomal RNA. Additional bands are the lower
marker, and the potentially 5S RNA. Presence of 5S RNA depends on the
purification method generally showing lower abundance in column purified
total RNA (see Figure 10).

One-Color Microarray-Based Gene Expression Analysis (Low Input Quick Amp Labeling) Protocol 75
3 Supplemental Procedures 
Step 2. Assess the quality using the Agilent 2100 Bioanalyzer

18S
28S

5S

LM

Figure 10 Analysis of (human) total RNA with the Eukaryote total RNA Nano assay using three different
samples with decreasing integrity: Red, RIN 8.4; Blue, RIN 5.9; Green, RIN 3,6. Characteristic
regions for ribosomal peaks and the lower marker (LM) are displayed.

Labeled cRNA
The resulting electropherogram should have a broad band. The majority of
signal for amplified sample should fall into the size range from 200 to 2000
nucleotides. If there isn't a band in this range, and there are distinct bands
less than 200 nucleotides in length, DO NOT proceed with that sample since it
has likely been degraded and will not provide accurate results. See Figure 11.

76 One-Color Microarray-Based Gene Expression Analysis (Low Input Quick Amp Labeling) Protocol
 Supplemental Procedures 3
Step 2. Assess the quality using the Agilent 2100 Bioanalyzer

LM

Figure 11 Smear analysis on non-fragmented Cy3 labeled cRNA allows determination of relative
concentration of products within size regions. For example, 52 % of overall RNA signal results from
fragments with size from 200 to 2000 nt.

For general assistance on evaluation of total RNA with emphasis on the RNA
integrity number, see the corresponding application note: “RNA integrity
number (RIN) - Standardization of RNA quality control”, 5989-1165EN.
Additional information on mRNA can be found in the corresponding
application notes: Interpreting mRNA electropherograms, publication
5988-3001EN, and Optimizing cRNA fragmentation for microarray
experiments using the Agilent 2100 bioanalyzer, publication 5988-3119EN.
To download application notes regarding the 2100 bioanalyzer visit Agilent
web site at [Link]/chem/labonachip.

One-Color Microarray-Based Gene Expression Analysis (Low Input Quick Amp Labeling) Protocol 77
3 Supplemental Procedures 
Step 3. Assess the quality using a NanoDrop Spectrophotometer

Step 3. Assess the quality using a NanoDrop Spectrophotometer

Accurate assessment of total RNA quantity and quality are crucial to the
success of an Agilent Gene Expression experiment. High quality RNA should
be free of contaminants such as carbohydrates, proteins, and traces of organic
solvents, and should also be intact with minimal degradation.
Use the NanoDrop ND-1000 UV-VIS Spectrophotometer (or equivalent) to
assess RNA concentration and purity.

UV-VIS Spectrophotometry
1 In the Nanodrop program menu, select Nucleic Acid Measurement, then
select Sample Type to be RNA-40.
2 Use 1.5 μL of nuclease-free water to blank the instrument.
3 Use 1.5 μL of each total RNA sample to measure RNA concentration. Record
the RNA concentration (ng/μL) for each sample.
4 Record the A260/A280 and A260/A230 ratios.
High-quality total RNA samples have an A260/A280 ratio of 1.8 to 2.0, which
indicates the absence of contaminating proteins. They also have an
A260/A230 ratio of >2.0, which indicates the absence of other organic
compounds, such as guanidinium isothiocyanate, alcohol and phenol as
well as cellular contaminants such as carbohydrates.
An A260/A230 ratio of <2.0 can indicate the presence of these contaminants,
which can interfere with the labeling reaction or can lead to inaccurate
quantification of your total RNA.

78 One-Color Microarray-Based Gene Expression Analysis (Low Input Quick Amp Labeling) Protocol
 Supplemental Procedures 3
Preventing Ozone-Related Problems

Preventing Ozone-Related Problems

The Agilent one-color platform is robust in environments where the ozone
level is 50 ppb (approximately 100 μg/m3) or less. Beyond this level, ozone can
significantly affect Cy3 signal and compromise microarray performance.
For Scanner C and Scanner B, the Agilent Ozone-Barrier Slide cover is
designed to protect against ozone-induced degradation of cyanine dyes and is
recommended when using Agilent oligo-based microarrays in high-ozone
environments. See step 14 on page 45.
For the Agilent SureScan scanner, two built-in mechanisms minimize dye
signal degradation by ozone and other dye oxidants:
• SureScan slide holder with an integrated ozone barrier in its lid.
• Catalytic ozone decomposition filtering system inside the scanner.
In addition to the ozone barriers, the Agilent Stabilization and Drying
Solution, which is an organic solvent based wash, can reduce background
variability produced by wash artifacts.
The use of the Agilent Stabilization and Drying Solution is described in this
section. For more information, visit
[Link]/chem/dnatechnicalnotes to download the technical note on
Improving Microarray Results by Preventing Ozone-Mediated Fluorescent
Signal Degradation (p/n 5989-0875EN).

One-Color Microarray-Based Gene Expression Analysis (Low Input Quick Amp Labeling) Protocol 79
3 Supplemental Procedures 
Step 1. Prepare the Stabilization and Drying Solution

Step 1. Prepare the Stabilization and Drying Solution

The Agilent Stabilization and Drying Solution contains an ozone scavenging
compound dissolved in acetonitrile. The compound in solution is present in
saturating amounts and may precipitate from the solution under normal
storage conditions. If the solution shows visible precipitation, warming of the
solution will be necessary to redissolve the compound. Washing slides using
Stabilization and Drying Solution showing visible precipitation will have a
profound adverse effect on microarray performance.

The Agilent Stabilization and Drying Solution is a flammable liquid. Warming the
WA R N I N G
solution will increase the generation of ignitable vapors.
Do not use an open flame or a microwave. Do not increase temperature rapidly.
Warm and mix the material away from ignition sources.
Use gloves and eye/face protection in every step of the warming procedures.

Failure to follow the outlined process will increase the potential for fire, explosion,
WA R N I N G
and possible personal injury. Agilent assumes no liability or responsibility for
damage or injury caused by individuals performing this process.

1 Warm the solution slowly in a water bath or a vented conventional oven at

40°C in a closed container with sufficient head space to allow for
expansion.

The original container can be used to warm the solution. Container volume is 700 mL and
NOTE contains 500 mL of liquid. If a different container is used, maintain or exceed this
headspace/liquid ratio. The time needed to completely redissolve the precipitate is
dependent on the amount of precipitate present, and may require overnight warming if
precipitation is heavy. DO NOT FILTER the Stabilization and Drying solution.

2 If needed, gently mix to obtain a homogeneous solution.

Mix under a vented fume hood away from open flames, or other sources of
ignition. Warm the solution only in a controlled and contained area that
meets local fire code requirements.
3 After the precipitate is completely dissolved, let the covered solution stand
at room temperature, allowing it to equilibrate to room temperature and
make sure that precipitation does not occur prior to use.

80 One-Color Microarray-Based Gene Expression Analysis (Low Input Quick Amp Labeling) Protocol
 Supplemental Procedures 3
Step 2. Wash with Stabilization and Drying Solution

Step 2. Wash with Stabilization and Drying Solution

Use fresh Gene Expression Wash Buffer for each wash group (up to eight slides). The
NOTE acetonitrile and Stabilization and Drying Solution can be reused for washing of up to three
groups of slides (for a total of 24 slides).

The Stabilization and Drying Solution must be set-up in a fume hood. Wash 1 and
WA R N I N G
Wash 2 set-up areas should be put close to, or preferably in, the same fume hood.
Use gloves and eye/face protection in every step of the warming procedures.

Table 31 lists the wash conditions for the wash procedure with Stabilization
and Drying Solution.

Table 31 Wash conditions

Dish Wash Buffer Temperature Time

Disassembly 1 Gene Expression Wash Buffer 1 Room temperature

1st wash 2 Gene Expression Wash Buffer 1 Room temperature 1 minute

2nd wash 3 Gene Expression Wash Buffer 2 Elevated 1 minute

temperature*

Acetonitrile Wash 4 acetonitrile Room temperature 10 seconds

3rd wash 5 Stabilization and Drying Solution Room temperature 30 seconds

* The elevated temperature of the second wash step is usually around 31°C due to cooling by the
room temperature dish and the rack of arrays.

1 Completely fill slide-staining dish #1 with Gene Expression Wash Buffer 1

at room temperature.
2 Put a slide rack into slide-staining dish #2. Add a magnetic stir bar. Fill
slide-staining dish #2 with enough Gene Expression Wash Buffer 1 at room
temperature to cover the slide rack. Put this dish on a magnetic stir plate.
3 Put the empty dish #3 on the stir plate and add a magnetic stir bar. Do not
add the pre-warmed (37°C) Gene Expression Wash Buffer 2 until the first
wash step has begun.

One-Color Microarray-Based Gene Expression Analysis (Low Input Quick Amp Labeling) Protocol 81
3 Supplemental Procedures 
Step 2. Wash with Stabilization and Drying Solution

4 Fill slide-staining dish #4 approximately three-fourths full with

acetonitrile. Add a magnetic stir bar and put this dish on a magnetic stir
plate.
5 Fill slide-staining dish #5 approximately three-fourths full with
Stabilization and Drying Solution. Add a magnetic stir bar and put this dish
on a magnetic stir plate.
6 Remove one hybridization chamber from incubator and record time. Record
whether bubbles formed during hybridization, and if all bubbles are
rotating freely.
7 Prepare the hybridization chamber disassembly.
a Put the hybridization chamber assembly on a flat surface and loosen the
thumbscrew, turning counter-clockwise.
b Slide off the clamp assembly and remove the chamber cover.
c With gloved fingers, remove the array-gasket sandwich from the chamber
base by grabbing the slides from their ends. Keep the microarray slide
numeric barcode facing up as you quickly transfer the sandwich to
slide-staining dish #1.
d Without letting go of the slides, submerge the array-gasket sandwich into
slide-staining dish #1 containing Gene Expression Wash Buffer 1.
8 With the sandwich completely submerged in
Gene Expression Wash Buffer 1, pry the sandwich open from the barcode
end only:
a Slip one of the blunt ends of the forceps between the slides.
b Gently turn the forceps upwards or downwards to separate the slides.
c Let the gasket slide drop to the bottom of the staining dish.
d Remove the microarray slide and put into slide rack in the slide-staining
dish #2 containing Gene Expression Wash Buffer 1 at room temperature.
Minimize exposure of the slide to air. Touch only the barcode portion of
the microarray slide or its edges!
9 Repeat step 6 through step 8 for up to seven additional slides in the group.
A maximum of eight disassembly procedures yielding eight microarray
slides is advised at one time in order to facilitate uniform washing.
10 When all slides in the group are put into the slide rack in slide-staining dish
#2, stir using setting 4 for 1 minute.
11 During this wash step, remove Gene Expression Wash Buffer 2 from the
37°C water bath and pour into the Wash 2 dish.

82 One-Color Microarray-Based Gene Expression Analysis (Low Input Quick Amp Labeling) Protocol
 Supplemental Procedures 3
Step 2. Wash with Stabilization and Drying Solution

12 Transfer slide rack to slide-staining dish #3 containing

Gene Expression Wash Buffer 2 at elevated temperature. Stir using setting
4 for 1 minute.
13 Remove the slide rack from Gene Expression Wash Buffer 2 and tilt the
rack slightly to minimize wash buffer carry-over. Immediately transfer the
slide rack to slide-staining dish #4 containing acetonitrile and stir using
setting 4 for less than 10 seconds.
14 Transfer the slide rack to dish #5 filled with Stabilization and Drying
Solution and stir using setting 4 for 30 seconds.
15 Slowly remove the slide rack trying to minimize droplets on the slides. It
should take 5 to 10 seconds to remove the slide rack.
16 Discard used Gene Expression Wash Buffer 1 and
Gene Expression Wash Buffer 2.
17 Repeat steps 1 through 16 for the next group of eight slides using fresh
Gene Expression Wash Buffer 1 and Gene Expression Wash Buffer 2
pre-warmed to 37°C.
18 Scan slides immediately to minimize the impact of environmental oxidants
on signal intensities. If necessary, store slides in orange slide boxes in a
nitrogen purge box, in the dark.

Dispose of acetonitrile and Stabilization and Drying Solution as flammable solvents.

CAUTION

19 Immediately continue at step 14 on page 45.

One-Color Microarray-Based Gene Expression Analysis (Low Input Quick Amp Labeling) Protocol 83
3 Supplemental Procedures 
Normalizing Agilent One-Color Microarray Data

Normalizing Agilent One-Color Microarray Data

When comparing data across a set of one-color microarrays, a simple linear
scaling of the data is usually sufficient for most experimental applications.
Agilent has determined that the signal value of the 75th percentile of all of
non-control probes on the microarray is a more robust and representative
value of the overall microarray signal as compared to the median or 50th
percentile signal. Therefore, use the 75th percentile signal value to normalize
Agilent one-color microarray signals for inter-array comparisons.

To do downstream analysis of Agilent microarray data

• Use GeneSpring GX 9.0 or later.
Note that the default normalization scheme for Agilent one-color data in the
GeneSpring GX 9.0 (or later) program is 75th percentile scaling.
For more information on the GeneSpring GX program, go to
[Link]

84 One-Color Microarray-Based Gene Expression Analysis (Low Input Quick Amp Labeling) Protocol
 Supplemental Procedures 3
To use Feature Extraction

To use Feature Extraction

To normalize Agilent one-color microarray data without the GeneSpring
program, use the 75th percentile value for each microarray assay in the
Agilent Feature Extraction text file.
1 Generate a Feature Extraction text file.
2 Find the “STATS Table” in the middle section of the text file. This section
describes the results from the array-wide statistical calculations.
3 Find the 75th percentile value of the non-control signals under the column
with the heading gPercentileIntensityProcessedSignal.
4 Divide each of the green processed signals (gProcessedSignal) by the 75th
percentile signal (gPercentileIntensityProcessedSignal) to generate
the 75th percentile normalized microarray processed signals.
You can further scale the resulting 75th percentile-normalized signals by a
constant, such as the average of the 75th percentile signals of the arrays in the
experiment.
For more information on the output from the Agilent Feature Extraction
program, please refer to the Agilent G2567AA Feature Extraction Software
Reference Guide. You can download this guide from the Agilent web site at
[Link]/chem/dnamanuals-protocols.

One-Color Microarray-Based Gene Expression Analysis (Low Input Quick Amp Labeling) Protocol 85
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86 One-Color Microarray-Based Gene Expression Analysis (Low Input Quick Amp Labeling) Protocol
 One-Color Microarray-Based Gene Expression Analysis
Protocol

4
Reference
Kit Contents 88
Supplemental User Guides 91
Microarray Handling Tips 92
General Microarray Layout and Orientation 93
Array/Sample tracking microarray slides 96
Related Microarray Reagents 98

This chapter contains reference information related to the protocol and

Feature Extraction default parameter settings

87
4 Reference 
Kit Contents

Kit Contents
The content of the kits used in this protocol (required and optional) are listed
here.

Table 32 Low Input Quick Amp Labeling Kit, One-Color

Content
T7 Primer (green cap)
5× First Strand Buffer (green cap)
0.1 M DTT (white cap)
10 mM dNTP Mix (green cap)
Affinity Script RNase Block Mix (violet cap)
5× Transcription Buffer (blue cap)
NTP Mix (blue cap)
T7 RNA Polymerase Blend (red cap)
Nuclease-free Water
Cyanine 3-CTP

Table 33 RNA Spike-In Kit, One-Color

Content
Spike Mix
Dilution Buffer

Table 34 Gene Expression Hybridization Kit

Content
10× Gene Expression Blocking Agent
25× Fragmentation Buffer
2× Hi-RPM Hybridization Buffer

88 One-Color Microarray-Based Gene Expression Analysis (Low Input Quick Amp Labeling) Protocol
 Reference 4
Kit Contents

Table 35 Gene Expression Wash Buffer Kit

Content
Gene Expression Wash Buffer 1
Gene Expression Wash Buffer 2
Triton X-102 (10%)

Table 36 RNeasy Mini Kit

Content
RNeasy Mini Spin Column (pink)
Collection Tube (1.5 ml)
Collection Tube (2 ml)
Buffer RLT
Buffer RW1
Buffer RPE
RNase-Free Water

Table 37 Absolutely RNA Nanoprep Kit

Content
Lysis Buffer
1.67× High Salt Wash Buffer
5× Low Salt Wash Buffer
Elution Buffer
DNase Reconstitution Buffer (green cap)
DNase Digestion Buffer (green cap)
Beta-Mercaptoethanol (yellow cap)
RNase-free DNase I
RNA-binding nano-spin cup

One-Color Microarray-Based Gene Expression Analysis (Low Input Quick Amp Labeling) Protocol 89
4 Reference 
Kit Contents

Table 37 Absolutely RNA Nanoprep Kit

Content
2-ml collection tube
RNA Binding Nano Spin Cup Cap

Table 38 Quick Amp Labeling Kit, One-Color

Content
T7 Promoter Primer (green cap)
5× First Strand Buffer (green cap)
0.1 M DTT (white cap)
10 mM dNTP Mix (green cap)
RNase Inhibitor (violet cap)
MMLV-RT (violet cap)
4× Transcription Buffer (clear cap)
NTP Mix (blue cap)
Inorganic Pyrophosphatase (red cap)
T7 RNA Polymerase Blend (red cap)
PEG (clear cap)
Cyanine 3-CTP

90 One-Color Microarray-Based Gene Expression Analysis (Low Input Quick Amp Labeling) Protocol
 Reference 4
Supplemental User Guides

Supplemental User Guides

First-time users of the Agilent oligo microarray system, please refer to the
following user manuals for detailed descriptions and operation
recommendations for each of the hardware and software components used in
the one-color platform workflow. The user guides can be downloaded from the
Agilent web site at [Link]/chem/dnamanuals-protocols.
• Agilent Microarray Hybridization Chamber User Guide
• Hybridization Oven User Manual
• Microarray Scanner System User Guide
• G4900DA SureScan Microarray Scanner User Guide
• Feature Extraction Software Quick Start Guide
• Feature Extraction Software User Guide
• Feature Extraction Software Reference Guide

One-Color Microarray-Based Gene Expression Analysis (Low Input Quick Amp Labeling) Protocol 91
4 Reference 
Microarray Handling Tips

Microarray Handling Tips

Each microarray is printed on the side of the glass slide containing the
“Agilent”-labeled barcode. This side is called the “active” side. The numeric
barcode is on the inactive side of the slide.

You must familiarize yourself with the assembly and disassembly instructions for use
CAUTION
with the Agilent Microarray Hybridization Chamber (G2534A) and gasket slides.
Practice slide kits are available.

In this “processing and hybridization” procedure, the hybridization mixture is

applied directly to the gasket slide, and not to the active side of the oligo
microarray. Instead, the active side of the oligo microarray is put on top of the
gasket slide to form a “sandwich slide” pair.
To avoid damaging the microarray, always handle glass slides carefully by
their edges. Wear powder-free gloves. Never touch the surfaces of the slides. If
you do, you may cause irreparable damage to the microarray.
Never allow the microarray surface to dry out during the hybridization
process and washing steps.

92 One-Color Microarray-Based Gene Expression Analysis (Low Input Quick Amp Labeling) Protocol
 Reference 4
General Microarray Layout and Orientation

General Microarray Layout and Orientation

Agilent oligo microarray (1 microarray/slide format) as imaged on the Agilent
microarray scanner

Microarrays are printed on the side of the glass labeled with the “Agilent” bar code
(also referenced as "active side" or "front side").
00116789

Agilent Microarray Agilent microarray slide holder for Scanner B

Scanner scans
through the glass.
and C (left) or SureScan microarray scanner
(Back side scanning.) (right)

Figure 12 Agilent microarray slide and slide holder. The opposite or “non-active”
numerically barcoded side is shown.

Agilent oligo microarray formats and the resulting “microarray design files”
are based on how the Agilent microarray scanner images 1-inch × 3-inch glass
slides. Agilent designed its microarray scanner to scan through the glass slide
(back side scanning). The glass slide is securely placed in an Agilent
microarray slide holder with the “Agilent” labeled barcode facing the opening
of the slide holder (on SureScan Microarray Scanner G4900DA) or facing the
inside of the slide holder (C scanner G2565CA). In this orientation, the “active
side” containing the microarrays is protected from potential damage by
fingerprints and other elements. Once securely placed, the numeric barcode,
non-active side of the slide, is visible from the outside of the slide holder.
Figure 12 depicts how the Agilent microarray scanner reads the microarrays
and how this relates to the “microarray design files” that Agilent generates
during the manufacturing process of its in situ-synthesized oligonucleotide
microarrays. Thus, if you have a scanner that reads microarrays from the
“front side” of the glass slide, the collection of microarray data points will be
different in relation to the “microarray design files” supplied with the Agilent
oligo microarray kit you purchased. Therefore, please take a moment to
become familiar with the microarray layouts for each of the Agilent oligo
microarrays and the layout information as it pertains to scanning using a
“front side” scanner.

One-Color Microarray-Based Gene Expression Analysis (Low Input Quick Amp Labeling) Protocol 93
4 Reference 
General Microarray Layout and Orientation

Non-Agilent front side microarray scanners

When imaging Agilent oligo microarray slides, you must determine:
• If the scanner images microarrays by reading them on the “front side” of the
glass slide (“Agilent”-labeled barcode side of the slide) and
• If the image produced by the non-Agilent scanner is oriented in a “portrait”
or “landscape” mode, “Agilent”-labeled barcode left-side, right-side, up or
down, as viewed as an image in the imaging software (see Figure 13).
This changes the feature numbering and location as it relates to the
“microarray design files” found on the CD in each Agilent oligo microarray kit.
Microarray layout maps are available from Agilent. For more information, go
to [Link]/chem/dnamanuals-protocols and download Agilent
Microarray Formats Technical Drawings with Tolerance (publication
G4502-90001). This document contains visual references and guides that will
help you determine the feature numbering as it pertains to your particular
scanner configuration.

94 One-Color Microarray-Based Gene Expression Analysis (Low Input Quick Amp Labeling) Protocol
 Reference 4
General Microarray Layout and Orientation

Front side
bar code up
(portrait)
Agilent

Agilent
Agilent

Front side Front side

bar code left bar code right
(landscape) (landscape)

Agilent

Front side
bar code down
(portrait)

Figure 13 Microarray slide orientation

One-Color Microarray-Based Gene Expression Analysis (Low Input Quick Amp Labeling) Protocol 95
4 Reference 
Array/Sample tracking microarray slides

Array/Sample tracking microarray slides

Use the forms below to make notes to track your samples on microarray slides.
Position the gasket slide in the SureHyb chamber base with the label to the
left. Load the samples: top row, left to right, then lower row, left to right. The
array suffix assignments from Feature Extraction will be in the order shown.

Arrays

Array 1_1 Array 1_2 Array 1_3 Array 1_4

Sample: Sample: Sample]ËËËËËËËËËËËËËËËËËËËËË.ample:

B
A
R
C
O
D
E

Barcode Number __________________________________________________________

Figure 14 4-pack microarray slides

96 One-Color Microarray-Based Gene Expression Analysis (Low Input Quick Amp Labeling) Protocol
 Reference 4
Array/Sample tracking microarray slides

Arrays

Array 1_1 Array 1_2 Array 1_3 Array 1_4

p
Sample: p
Sample: p
Sample]ËËËËËËËËËËËËËËËËËËËËË.ample:

B
A
R
C
O Sample: Sample: Sample]ËËËËËËËËËËËËËËËËËËËËË.ample:
D
E

A
Array 2 1
2_1 A
Array 2
2_2
2 A
Array 2_3
2 3 A
Array 2
2_4
4

Barcode Number __________________________________________________________

Figure 15 8-pack microarray slide

One-Color Microarray-Based Gene Expression Analysis (Low Input Quick Amp Labeling) Protocol 97
4 Reference 
Related Microarray Reagents

Related Microarray Reagents

Description Vendor and part number

Universal Human Reference RNA Agilent p/n 740000

Universal Mouse Reference RNA Agilent p/n 740100

Universal Rat Reference RNA Agilent p/n 740200

98 One-Color Microarray-Based Gene Expression Analysis (Low Input Quick Amp Labeling) Protocol
[Link]

In This Book
This guide contains
information to run the
One- Color
Microarray- Based Gene
Expression Analysis
protocol.

PR7000-0032
© Agilent Technologies, Inc. 2007-2015

Version 6.9.1, December 2015

*G4140-90040*
G4140-90040
Revision B6

Agilent Technologies