miRNA Microarray System with miRNA Complete

Labeling and Hyb Kit

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

Version 4.1, October 2021

Microarrays manufactured with Agilent SurePrint Technology

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changed, without notice, in future
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 In this Guide...
This document describes the Agilent recommended procedures for the
preparation and labeling of complex biological targets and hybridization, washing,
scanning, and feature extraction of Agilent oligonucleotide microarrays for
microarray-based miRNA systems.

1 Before You Begin

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

2 Procedures
This chapter describes the steps to prepare Spike-In solutions (optional), samples,
hybridize, wash and scan miRNA microarrays, and to extract data using the
Feature Extraction Software.

3 Supplemental Procedures
This chapter contains instructions for characterization of total RNA, and steps to
prevent ozone-related problems.

4 Reference
This chapter contains reference information related to the protocol and the limited
use license.

miRNA Microarray System with miRNA Complete Labeling and Hyb Kit Protocol 3
 What’s new in 4.1
• Clarification of sample storage temperature on page 32.

What’s new in 4.0

• Updated document look and feel.
• Instructions on handling the newly redesigned “secure fit” slide boxes in which
the microarray slides are shipped. Before opening the box for the first time, see
““Secure Fit” Slide Box Opening Instructions" on page 68.
• Expanded instructions and new images in the Procedures chapter to help avoid
common problems and optimize hybridization of your sample to the
microarray.
• Updated web addresses for Agilent materials.
• Removed microarray scanning instructions for the Agilent B scanner.
• Replaced detailed Safety Notes with guidance on consulting the safety data
sheets and product hazard labels.

What’s New in 3.1

• Corrected CIP Master Mix table.
• Updated product labeling statement.

4 miRNA Microarray System with miRNA Complete Labeling and Hyb Kit Protocol
 Content

1 Before You Begin 7

Procedural Notes 8
Safety Notes 9
Agilent Oligo Microarrays 9
Required Equipment 11
Required Reagents 13
Optional Equipment/Reagents 14
Required Hardware and Software 15
Optional Software 15
RNA Extraction Recommendation 15

2 Procedures 17
Sample Preparation and Labeling 19
Step 1. Prepare Spike-In solutions (optional) 19
Step 2. Dephosphorylate the sample 21
Step 3. Denature the sample 22
Step 4. Ligate the sample 23
Step 5. Purify the labeled RNA (optional) 24
Step 6. Dry the sample 26
Microarray Hybridization 27
Step 1. Prepare the 10× Blocking Agent 27
Step 2. Prepare hybridization samples 28
Step 3. Prepare the hybridization assembly 29
Step 4. Hybridize 36
Microarray Wash 37
Step 1. Add Triton X-102 to Gene Expression wash buffers 37
Step 2. Prewarm Gene Expression Wash Buffer 2 and slide-staining dish
#3 38
Step 3. Prepare the equipment 38
Step 4. Wash the microarray slides 39
Step 5. Put slides in a slide holder 43

miRNA Microarray System with miRNA Complete Labeling and Hyb Kit Protocol 5
 Content

Scanning and Feature Extraction 46

Step 1. Scan the slides 46
Step 2. Extract data using Agilent Feature Extraction Software 49

3 Supplemental Procedures 57
Normalizing Agilent One-Color Microarray Data 58
To do downstream analysis of Agilent microarray data 58
To use Feature Extraction 59
Characterization of Total RNA 60
Step 1. Prepare for characterization of total RNA 61
Step 2. Characterize the RNA samples using the Agilent 2100
Bioanalyzer 62

4 Reference 65
Kit Contents 66
Supplemental User Guides 67
“Secure Fit” Slide Box Opening Instructions 68
Microarray Handling Tips 70
General Microarray Layout and Orientation 71
Array/Sample tracking microarray slides 74

6 miRNA Microarray System with miRNA Complete Labeling and Hyb Kit Protocol
1 Before You Begin
Procedural Notes 8
Safety Notes 9
Agilent Oligo Microarrays 9
Required Equipment 11
Required Reagents 13
Optional Equipment/Reagents 14
Required Hardware and Software 15
Optional Software 15
RNA Extraction Recommendation 15

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

N OTE technical support to those who use non-Agilent protocols in processing Agilent
microarrays.

miRNA Microarray System with miRNA Complete Labeling and Hyb Kit Protocol 7
1 Before You Begin
Procedural Notes

Procedural Notes

• Determine the integrity 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.
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.
• In general, follow Biosafety Level 1 (BL1) safety rules.

8 miRNA Microarray System with miRNA Complete Labeling and Hyb Kit Protocol
1 Before You Begin
Safety Notes

Safety Notes

Wear appropriate personal protective equipment (PPE) when working in the

CAU TI O N laboratory.
For reagent safety information, consult the safety data sheets and any product
hazard labels. Agilent safety data sheets are available at [Link].

Agilent Oligo Microarrays

To get design files or create a custom design for any species in the miRBASE, go
to the Agilent eArray web site at [Link]

Store entire kit at room temperature. After breaking foil on microarray pouch,
N OTE store 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.

Eight microarrays are printed on each 1-inch × 3-inch glass slide

Catalog SurePrint HD and G3 Microarrays and Microarray Kits

Table 1 Catalog SurePrint HD and G3 miRNA Microarrays - Human

Part Number Description miRBase

Release

G4870C G3 Human miRNA Microarray Kit, Release 21, 8×60K (3 slides) 21.0

G4872A-070156 G3 Human miRNA Microarray, Release 21, 8×60K (1 slide) 21.0

G4872A-046064 G3 Human miRNA Microarray, Release 19.0, 8×60K (1 slide) 19.0

G4870A G3 Human miRNA Microarray Kit Release 16.0, 8×60K (3 slides) 16.0

G4872A-031181 G3 Human miRNA Microarray, Release 16.0, 8×60K (1 slide) 16.0

G4471A-029297 HD Human miRNA Microarray, Release 14.0, 8×15K (1 slide) 14.0

G4470C HD Human miRNA Microarray Kit (V3), 8×15K (3 slides) 12.0

G4471A-021827 HD Human miRNA Microarray (V3), 8×15K (1 slide) 12.0

miRNA Microarray System with miRNA Complete Labeling and Hyb Kit Protocol 9
1 Before You Begin
Agilent Oligo Microarrays

Table 1 Catalog SurePrint HD and G3 miRNA Microarrays - Human (continued)

Part Number Description miRBase

Release

G4470B HD Human miRNA Microarray (V2) Kit, 8×15K (3 slides) 10.1

G4471A-019118 HD Human miRNA Microarray (V2), 8×15K (1 slide) 10.1

G4470A HD Human miRNA Microarray (V1) Kit, 8×15K (3 slides) 9.1

G4471A-016436 HD Human miRNA Microarray (V1), 8×15K (1 slide) 9.1

Table 2 Catalog SurePrint G3 and HD miRNA Microarrays - Mouse

Part Number Description miRBase

Release

G4859C G3 Mouse miRNA Microarray Kit, Release 21, 8×60K (3 slides) 21.0

G4872A-070155 G3 Mouse miRNA Microarray, Release 21, 8×60K (1 slide) 21.0

G4872A-046065 G3 Mouse miRNA Microarray, Release 19.0, 8×60K (1 slide) 19.0

G4872A-038112 G3 Mouse miRNA Microarray, Release 18.0, 8×60K (1 slide) 18.0

G4859B G3 Mouse miRNA Microarray Kit, Release 17.0, 8×60K (3 slides) 17.0

G4872A-035430 G3 Mouse miRNA Microarray, Release 17.0, 8×60K (1 slide) 17.0

G4859A G3 Mouse miRNA Microarray Kit Release 16.0, 8×60K (3 slides) 16.0

G4872A-031184 G3 Mouse miRNA Microarray, Release 16.0, 8×60K (1 slide) 16.0

G4471A-029152 HD Mouse miRNA Microarray, Release 15.0, 8×15K (1 slide) 15.0

G4471A-021828 HD Mouse miRNA Microarray, Release 12.0, 8×15K (1 slide) 12.0

G4472B HD Mouse miRNA Microarray Kit (V2), 8×15K (3 slides) 12.0

G4472A HD Mouse miRNA Microarray Kit, 8×15K (3 slides) 10.1

G4471A-019119 HD Mouse miRNA Microarray, 8×15K (1 slide) 10.1

10 miRNA Microarray System with miRNA Complete Labeling and Hyb Kit Protocol
1 Before You Begin
Required Equipment

Table 3 Catalog SurePrint G3 and HD miRNA Microarrays - Rat

Part Number Description miRase

Release

G4473C HD Rat miRNA Microarray Kit, Release 21, 8×15K (3 slides) 21.0

G4471A-070154 HD Rat miRNA Microarray, Release 21, 8×15K (1 slide) 21.0

G4471A-046066 HD Rat miRNA Microarray, Release 19.0, 8×15K (1 slide) 19.0

G4473B HD Rat miRNA Microarray Kit Release 16.0, 8×15K (3 slides) 16.0

G4471A-031189 HD Rat miRNA Microarray, Release 16.0, 8×15K (1 slide) 16.0

G4471A-029200 HD Rat miRNA Microarray, Release 15.0, 8×15K (1 slide) 15.0

G4473A HD Rat miRNA Microarray Kit V1, 8×15K (3 slides) 10.1

G4471A-019159 HD Rat miRNA Microarray Slide V1, 8×15K (1 slide) 10.1

Custom Microarrays
Table 4 Custom SurePrint Microarray

Part Number Description

G4871A Custom G3 miRNA Microarray, 8×60K

G4474A Custom HD miRNA Microarray, 8×15K

Required Equipment

Table 5 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 Chamber gasket slides

8 microarrays/slide, 5 slides/box Agilent p/n G2534-60014
Go to [Link] to see all available
kit configurations.

Hybridization oven; temperature set at 55°C Agilent p/n G2545A

miRNA Microarray System with miRNA Complete Labeling and Hyb Kit Protocol 11
1 Before You Begin
Required Equipment

Table 5 Required Equipment (continued)

Description Vendor and part number

Hybridization oven rotator for Agilent Agilent p/n G2530-60029

Microarray Hybridization Chambers
nuclease-free 1.5 mL microfuge tube Thermo Fisher Scientific p/n AM12400 or
equivalent

magnetic stir bar (×2) Corning p/n 401435 or equivalent

magnetic stir plate Corning p/n 6795-410 or equivalent

magnetic stir plate with heating element Corning p/n 6795-420D or equivalent

microcentrifuge Eppendorf p/n 5417R or equivalent

sterile storage bottle Nalgene 455-1000 or equivalent

micropipettor Pipetman P-10, P-20, P-200, P-1000 or

equivalent

1.5L glass dish Pyrex p/n 7211 or equivalent

slide-staining dish, with slide rack (×3) Thermo Fisher Scientific p/n 121 or equivalent

vacuum concentrator with heater Savant SpeedVac p/n SPD111V or equivalent

circulating water baths or heat blocks set to

16°, 37°C, and 100°C,

clean forceps

ice bucket

powder-free gloves

sterile, nuclease-free aerosol barrier pipette

tips

vortex mixer

timer

nitrogen purge box for slide storage

12 miRNA Microarray System with miRNA Complete Labeling and Hyb Kit Protocol
1 Before You Begin
Required Reagents

Required Reagents

Table 6 Required Reagents

Description Vendor and part or catalog number

miRNA Complete Labeling and Hyb Kit Agilent p/n 5190-0456

Gene Expression Wash Buffer Kit Agilent p/n 5188-5327

DNase/RNase-free distilled water Thermo Fisher Scientific p/n

10977-015

Milli-Q water or equivalent

isopropyl alcohol (molecular biology grade)

miRNA Microarray System with miRNA Complete Labeling and Hyb Kit Protocol 13
1 Before You Begin
Optional Equipment/Reagents

Optional Equipment/Reagents

Table 7 Optional Equipment/Reagents

Description Vendor and part number

Universal miRNA Reference Sample Agilent p/n 750700

2100 Bioanalyzer Agilent p/n G2939AA

RNA 6000 Nano Assay Kit (RNA Series II Kit) Agilent p/n 5067-1511

RNA 6000 Pico Kit Agilent p/n 5067-1513

Stabilization and Drying Solution* Agilent p/n 5185-5979

Ozone-Barrier Slide Cover* Agilent p/n G2505-60550

Small RNA Kit Agilent p/n 5067-1548

microRNA Spike-In Kit Agilent p/n 5190-1934

Micro Bio-Spin P-6 Gel Column Bio-Rad p/n 732-6221

slide box Corning p/n 07201629

PCR 96 plate Eppendorf p/n 951020401

acetonitrile Sigma p/n 271004-1L

dye filter Sigma p/n Z361569

thermal cycler

1× tris EDTA pH 7.5

* Recommended when processing microarrays in high ozone environment.

14 miRNA Microarray System with miRNA Complete Labeling and Hyb Kit Protocol
1 Before You Begin
Required Hardware and Software

Required Hardware and Software

Table 8 Required Hardware and Software

Description

Feature Extraction software 9.5 or later without Spike-In or Feature Extraction 10.7.3 with Spike-In

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.

Optional Software

Table 9 Optional Software

Description

GeneSpring 10.0 or higher

RNA Extraction Recommendation

Total RNA extraction methods differ in numerous ways and may impact:
• yield
• the inclusion of small RNAs in the total RNA extraction
• the quantification of the total RNA
The Agilent miRNA Microarray system is for use with total RNA extracts. Do not
use size fractionation or small RNA enrichment protocols. The following method
has been validated on the Agilent miRNA Microarray system:
• Absolutely RNA miRNA Kit (Agilent p/n 400814)
To get consistent results for comparative experiments, use the same total RNA
extraction method. Different total RNA extraction methods can result in slightly
different miRNA profiles.

miRNA Microarray System with miRNA Complete Labeling and Hyb Kit Protocol 15
1 Before You Begin
RNA Extraction Recommendation

Do not use purified miRNA. This protocol supports the use of total RNA only.
CAU TI O N
Extraction methods that use organic solvents such as TRIZOL can result in
inaccurate quantification, because organic solvent contamination from
carry-over during the RNA extraction can compress the 260/230 ratio. The
affected 260 measurement can result in inaccurate quantification of the total
RNA. To ensure the removal of residual TRIZOL reagent, do one to two
additional chloroform extractions after the final extraction step in the TRIZOL
protocol, before you continue to the ethanol precipitation. See
“Characterization of Total RNA” on page 60 for more information on RNA
purity and quality assessment.

16 miRNA Microarray System with miRNA Complete Labeling and Hyb Kit Protocol
2 Procedures
Sample Preparation and Labeling 19
Step 1. Prepare Spike-In solutions (optional) 19
Step 2. Dephosphorylate the sample 21
Step 3. Denature the sample 22
Step 4. Ligate the sample 23
Step 5. Purify the labeled RNA (optional) 24
Step 6. Dry the sample 26
Microarray Hybridization 27
Step 1. Prepare the 10× Blocking Agent 27
Step 2. Prepare hybridization samples 28
Step 3. Prepare the hybridization assembly 29
Step 4. Hybridize 36
Microarray Wash 37
Step 1. Add Triton X-102 to Gene Expression wash buffers 37
Step 2. Prewarm Gene Expression Wash Buffer 2 and slide-staining dish
#3 38
Step 3. Prepare the equipment 38
Step 4. Wash the microarray slides 39
Step 5. Put slides in a slide holder 43
Scanning and Feature Extraction 46
Step 1. Scan the slides 46
Step 2. Extract data using Agilent Feature Extraction Software 49

The Agilent miRNA Microarray System uses cyanine 3-labeled targets to

measure miRNA in experimental and control samples. Figure 1 is a standard
workflow for sample preparation and array hybridization.

miRNA Microarray System with miRNA Complete Labeling and Hyb Kit Protocol 17
2 Procedures

Figure 1. Workflow for sample preparation and array processing with the optional Bio-Rad columns and spike-ins.

18 miRNA Microarray System with miRNA Complete Labeling and Hyb Kit Protocol
2 Procedures
Sample Preparation and Labeling

Sample Preparation and Labeling

The miRNA Complete Labeling and Hyb Kit generates fluorescently-labeled

miRNA with a sample input of 100 ng of total RNA. This method involves the
ligation of one Cyanine 3-pCp molecule to the 3' end of a RNA molecule with
greater than 90% efficiency. The miRNA Complete Labeling and Hyb Kit provides
all the needed components for sample labeling and hybridization preparation.
See Table on page 66.

Please refer to “Characterization of Total RNA” on page 60 for procedural

N OTE recommendations on total RNA characterization.
Read the protocol in its entirety before you continue.

This protocol is compatible with hybridization of labeled miRNA to either 8×15K

or 8×60K microarray formats.

Step 1. Prepare Spike-In solutions (optional)

The microRNA Spike-In Kit consists of two microRNA Spike-In solutions for
process control. Use the Spike-In solutions to help distinguish significant
biological data from processing issues. The Labeling Spike-In solution is used in
the labeling reaction while the Hyb Spike-In solution is used in the hybridization
reaction.

Do not use spike-in solutions for normalization purposes.

CAU TI O N
Do not use with drosophila miRNA microarrays. Spike-in solutions are derived
from drosophila sequences.

Prepare the 2nd Dilution Spike-In solutions and the 3rd Dilution Spike-In
N OTE solutions just prior to use. If 1st Dilution Spike-In solutions is already prepared,
start at step 7.

miRNA Microarray System with miRNA Complete Labeling and Hyb Kit Protocol 19
2 Procedures
Step 1. Prepare Spike-In solutions (optional)

1 Thaw all components.

2 Briefly spin tubes.
3 Label 10 reaction tubes:
• Label 5 tubes “1st Dilution Labeling Spike-In”
• Label 5 tubes “1st Dilution Hyb Spike-In”.
4 In each tube, add the components in Table 10 according to label on the tube.
Table 10 1st Dilution Spike-In solutions

1st Dilution Labeling Spike-In solution 1st Dilution Hyb Spike-In solution

198 μL of the Dilution Buffer 198 μL of the Dilution Buffer

2 μL of the Labeling Spike-In solution 2 μL of the Hyb Spike-In solution

5 Mix well and briefly spin tubes.

6 (Optional) To increase the number of uses of this kit, and to prevent excess
freeze/thaw cycles, store small aliquots of the unused 1st Dilution Spike-In
solutions in labeled tubes at -80°C.
7 Label one new tube each with these labels (total of 4 tubes):
• “2nd Dilution Labeling Spike-In”
• “3rd Dilution Labeling Spike-In”
• “2nd Dilution Hyb Spike-In”
• “3rd Dilution Hyb Spike-In”
8 Make the 2nd Dilution Spike-In solutions:
• Into each of the tubes that are labeled for 2nd Dilution, add the contents in
Table 11 according to label on the tube.
Table 11 2nd Dilution Spike-In solutions

2nd Dilution Labeling Spike-In solution 2nd Dilution Hyb Spike-In solution

198 μL of nuclease-free water. 198 μL of nuclease-free water.

2 μL of 1st Dilution Labeling Spike-In solution 2 μL of 1st Dilution Hyb Spike-In solution

9 Mix well and briefly spin in a centrifuge.

20 miRNA Microarray System with miRNA Complete Labeling and Hyb Kit Protocol
2 Procedures
Step 2. Dephosphorylate the sample

10 Make the 3rd Dilution Spike-In solutions:

• Into each of the tubes that are labeled for 3rd Dilution, add the contents in
Table 12 according to the label on the tube.
Table 12 3rd Dilution Spike-In solutions

3rd Dilution Labeling Spike-In solution 3rd Dilution Hyb Spike-In solution

198 μL of nuclease-free water. 198 μL of nuclease-free water.

2 μL of 2nd Dilution Labeling Spike-In solution 2 μL of 2nd Dilution Hyb Spike-In solution

11 Mix well and briefly spin in a centrifuge.

This dilution is used in the labeling reaction or the hybridization reaction.
12 Discard the 2nd Dilution Spike-In solutions and 3rd Dilution Spike-In
solutions after use.
13 Store 1st Dilution Spike-In solutions at -80°C.

Do not freeze/thaw the original or 1st Dilution Spike-In solutions more than
CAU TI O N
twice.

Step 2. Dephosphorylate the sample

1 Dilute total RNA sample to 50 ng/μL in 1× tris EDTA pH 7.5 or

DNase/RNase-free distilled water.
2 Add 2 μL (100 ng) of the diluted total RNA to a nuclease-free 1.5 mL
microfuge tube and maintain on ice.
3 Immediately prior to use, add the components in the order indicated in
Table 13 to create the CIP Master Mix. Maintain on ice.
If you did not prepare 3rd Dilution Labeling Spike-In solution, use
nuclease-free water.

miRNA Microarray System with miRNA Complete Labeling and Hyb Kit Protocol 21
2 Procedures
Step 3. Denature the sample

Table 13 CIP Master Mix

Components Volume (μL) per Volume (μL) per 9

reaction reactions

10× Calf Intestinal Phosphatase Buffer 0.4 3.6

3rd Dilution Labeling Spike-In solution or 1.1 9.9

nuclease-free water

Calf Intestinal Phosphatase 0.5 4.5

Total Volume 2.0 18.0

4 Add 2 μL of the CIP Master Mix to each sample tube for a total reaction
volume of 4 μL. Gently mix by pipetting.
5 Incubate the reaction at 37°C in a circulating water bath or heat block for 30
minutes to dephosphorylate the sample.
If needed, the samples can be stored at -80°C after incubation.

A thermal cycler, in conjunction with PCR plates, can be used to process a large
N OTE number of samples. Use a PCR cap to seal the plates. Do not use tape.

Step 3. Denature the sample

1 Add 2.8 μL of DMSO to each sample.

2 Incubate samples at 100°C in a circulating water bath or heat block for 5 to 10
minutes.

Incubate the sample for no less than 5 minutes and no more than 10 minutes,
CAU TI O N
or the labeling efficiency of the sample will be affected.

3 Immediately transfer to ice-water bath.

22 miRNA Microarray System with miRNA Complete Labeling and Hyb Kit Protocol
2 Procedures
Step 4. Ligate the sample

You must use an ice-water bath and not just crushed ice to ensure that the
CAU TI O N samples remain properly denatured.
When using a thermal cycler, transfer the PCR plate to an ice-water bath
immediately after incubating at 100°C to prevent the RNA from re-annealing.
Thermocyclers do not cool as quickly as is needed.

4 Continue to the next step immediately.

Step 4. Ligate the sample

1 Warm the 10× T4 RNA Ligase Buffer at 37°C and mix on a vortex mixer until
all precipitate is dissolved.

Make sure that the 10× T4 RNA Ligase Buffer has cooled to room temperature
CAU TI O N
before you proceed. Failure to do so will affect the T4 RNA Ligase activity, and
thus the labeling efficiency.

The Cyanine 3-pCp is light-sensitive. Please minimize any exposure to light.

CAU TI O N

2 Immediately prior to use, prepare the Ligation Master Mix. Gently mix the
components listed in Table 14 and maintain on ice.
Table 14 Ligation Master Mix

Components Volume (μL) per reaction Volume (μL) per 9 reactions

10× T4 RNA Ligase Buffer 1.0 9.0

Cyanine 3-pCp 3.0 27.0

T4 RNA Ligase 0.5 4.5

Total Volume 4.5 40.5

miRNA Microarray System with miRNA Complete Labeling and Hyb Kit Protocol 23
2 Procedures
Step 5. Purify the labeled RNA (optional)

Be sure to use the Ligation Master Mix within 15 minutes of mixing all the
CAU TI O N
components in Table 14. Failure to do so may affect the labeling efficiency.

3 Immediately add 4.5 μL of the Ligation Master Mix to each sample tube for a
total reaction volume of 11.3 μL.
4 Gently mix by pipetting and gently spin down.
5 Incubate at 16°C in a circulating water bath or cool block for 2 hours.
The sample can be stored at -80°C, if needed.

Step 5. Purify the labeled RNA (optional)

If you are not using the columns for purification, continue to “Step 6. Dry the
sample” on page 26.
This step removes DMSO and free Cyanine 3-pCp. The use of the column
reduces the needed drying time in “Step 6. Dry the sample”, but it adds hands-on
processing time.
Use the Micro Bio-Spin P-6 Gel Column.

Preparation of Micro Bio-Spin P-6 Gel Column

1 Invert the Micro Bio-Spin P-6 Gel Column sharply several times to resuspend
the settled gel and to remove any air bubbles.
2 Snap off the tip and place into a 2-mL collection tube supplied with the Micro
Bio-Spin P-6 Gel Column.
3 Remove the green cap from the Micro Bio-Spin P-6 Gel Column. If the buffer
does not drip into the 2-mL collection tube, press the green cap back onto the
Micro Bio-Spin P-6 Gel Column and remove it again. Let the buffer drain for
about 2 minutes.
4 Check to make sure that all columns are evenly drained.
5 Discard the drained buffer from the 2-mL collection tube and then place the
Micro Bio-Spin P-6 Gel Column back into the tube.
6 Spin the microcentrifuge tube containing the Micro Bio-Spin P-6 Gel Column
for 2 minutes at 1000 × g in a centrifuge.

24 miRNA Microarray System with miRNA Complete Labeling and Hyb Kit Protocol
2 Procedures
Step 5. Purify the labeled RNA (optional)

The speed of the microcentrifuge must be accurately set (for example,

CAU TI O N
1000 x g, not 1000 rpm). If the spin speed is too low, sample can be lost. If the
spin speed is too high, desalting may be ineffective and the column may break
down.

7 Remove the Micro Bio-Spin P-6 Gel Column from the 2-mL collection tube
and place it into a clean nuclease-free 1.5 mL microfuge tube. Discard the
2-mL collection tube.
The sample can be stored at -80°C, if needed.

Sample purification
1 Add 38.7 μL of 1× tris EDTA pH 7.5 or DNase/RNase-free distilled water to
the labeled sample for a total volume of 50 μL.
2 Without disturbing the gel bed, use a pipette to transfer the 50 μL sample onto
the gel bed from step 7 above.
3 Spin the microcentrifuge tubes that contain the columns for 4 minutes at
1000 × g in a centrifuge to elute the purified sample.
4 Discard the columns and keep on ice the flow-through that contains miRNA
sample.
5 Check that the final flow-through is translucent and slightly pink. The
flow-through volume needs to be uniform across the samples and close to
50 μL.

Do not discard the final flow-trough. It now contains the labeled miRNA
CAU TI O N
sample.

The sample can be stored at -80°C, if needed.

miRNA Microarray System with miRNA Complete Labeling and Hyb Kit Protocol 25
2 Procedures
Step 6. Dry the sample

Step 6. Dry the sample

1 After the 16°C labeling reaction or sample purification, completely dry the
samples. Use a vacuum concentrator with heater at 45 to 55°C or on the
medium-high heat setting. This step can take up to 1 hour after column
purification and up to 3 hours without column purification.
2 Check the samples after 30 minutes. Continue vacuum concentration until
they are dry.
To check for sample dryness, flick hard on the tube and make sure that the
pellets do not move or spread.

The sample must be completely dried after labeling. Residual DMSO will
CAU TI O N
adversely affect the hybridization results.

The sample can be stored at -80°C, if needed.

26 miRNA Microarray System with miRNA Complete Labeling and Hyb Kit Protocol
2 Procedures
Microarray Hybridization

Microarray Hybridization

Before you begin, make sure you read and understand ““Secure Fit” Slide Box
Opening Instructions” on page 68 and “Microarray Handling Tips” on page 70.
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
CAU TI O N
collected data. Refer to Agilent G2545A Hybridization Calibration Procedure
(publication G2545-90002, version A1 or higher) for more information.

Step 1. Prepare the 10× Blocking Agent

1 Add 125 μL of nuclease-free water to the vial containing lyophilized 10× Gene
Expression Blocking Agent supplied with the miRNA Complete Labeling and
Hyb Kit.
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.

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

miRNA Microarray System with miRNA Complete Labeling and Hyb Kit Protocol 27
2 Procedures
Step 2. Prepare hybridization samples

Step 2. Prepare hybridization samples

1 Equilibrate water bath or heat block to 100°C.

2 Resuspend the dried sample in 17 μL of nuclease-free water when the Hyb
Spike-In solution is used and 18 μL when the Hyb Spike-In solution is not
used.
3 For each microarray, add each of the components as indicated in Table 15 or
Table 16 to a 1.5 mL nuclease-free microfuge tube:
4 Mix well but gently on a vortex mixer.
Table 15 Hybridization mix for miRNA microarrays with Hyb Spike-In solution

Components Volume (μL)

Labeled miRNA sample 17

3rd Dilution Hyb Spike-In solution 1

10× Gene Expression Blocking Agent 4.5

2× Hi-RPM Hybridization Buffer 22.5

Total Volume 45

Table 16 Hybridization mix for miRNA microarrays without Hyb Spike-In solution

Components Volume (μL)

Labeled miRNA sample 18

10× Gene Expression Blocking Agent 4.5

2× Hi-RPM Hybridization Buffer 22.5

Total Volume 45

For best results, stagger the tubes in sets of eight such that eight arrays on
one array slide can be loaded at once. Have the SureHyb chamber along with
the gaskets and microarrays easily available to prepare the hybridization
assembly before you continue to the next step.
5 Incubate at 100°C for 5 minutes.
6 Immediately transfer to an ice water bath for 5 minutes.

28 miRNA Microarray System with miRNA Complete Labeling and Hyb Kit Protocol
2 Procedures
Step 3. Prepare the hybridization assembly

Do not leave samples in the ice water bath for more than 15 minutes. Longer
CAU TI O N
incubations may adversely affect the hybridization results.

7 Quickly spin in a centrifuge to collect any condensation at the bottom of the

tube.
8 Immediately proceed to “Step 3. Prepare the hybridization assembly” on
page 29.

Step 3. Prepare the hybridization assembly

Refer to the Agilent Microarray Hybridization Chamber User Guide (publication

G2534-90004) for in-depth instructions on how to load samples, assemble and
disassemble chambers, as well as other helpful tips. This user guide can be
downloaded from the Agilent Web site at [Link].
Before you begin, make sure you read and understand ““Secure Fit” Slide Box
Opening Instructions” on page 68 and “Microarray Handling Tips” on page 70.

Remove gasket slide from its packaging

• Do not remove gasket slide from protective sleeve until ready for use.
N OTE
• Do not slice or cut open the gasket slide protective packaging.
• Handle only the edges of the gasket slide.
• Prior to use, inspect gasket slides for visible gaps or cuts through the gaskets
or any debris within the hybridization areas as these are indications of
instability. Do not use gasket slides that have these features.

miRNA Microarray System with miRNA Complete Labeling and Hyb Kit Protocol 29
2 Procedures
Step 3. Prepare the hybridization assembly

1 With tweezers, carefully lift up the corner of the clear plastic covering and
slowly pull back the protective film.

Figure 2. Removal of clear plastic covering

2 With clean, powder-free gloved fingers, remove the gasket slide from its
package. Handle the slide only on its edges.
To avoid any potential contamination from surrounding surface materials,
immediately insert the gasket slide in the chamber base using the instructions
below.

Insert the gasket slide into the chamber base

1 Hold the gasket slide so that the barcode label is facing towards you. This
side of the slide is the gasket side.
2 Locate the four chamber base guideposts and rectangular barcode guide in
the chamber base.
3 Position the gasket slide between the 4 chamber base guide posts with the
barcode label resting over the base’s rectangular barcode guide.
Figure 3 shows the proper positioning using a 4-well gasket slide. Note,
however, that your gasket slide will contain 8 wells rather than 4.

30 miRNA Microarray System with miRNA Complete Labeling and Hyb Kit Protocol
2 Procedures
Step 3. Prepare the hybridization assembly

Figure 3. Chamber base, guide posts denoted with arrows

4 Gently place the gasket slide into the chamber base.

5 Make sure the gasket slide rests flush against the chamber base. Re-adjust to
a flush position against the chamber base if needed.

Slide and gasket are flush

Figure 4. Correct positioning of gasket slide in chamber base

Load the sample

1 Slowly dispense the entire volume of a hybridization sample mixture onto a
gasket well in a “drag and dispense” manner (described below). Load all 8
gasket wells before you add the microarray slide.
• Load the samples left to right, starting with the first row. The output files
will come out in that same order. Refer to “Array/Sample tracking
microarray slides” on page 74 for guidelines on tracking sample position
for multipack slide formats.

miRNA Microarray System with miRNA Complete Labeling and Hyb Kit Protocol 31
2 Procedures
Step 3. Prepare the hybridization assembly

The “drag and dispense” method helps to distribute the sample evenly across
the surface of the well and avoids spillover of sample over the gasket edge.
Start with the pipette tip near the top edge of the well. Do not directly touch the
gasket or the glass with the pipette tip. Then, dispense the mixture while you
move your pipette tip to the opposite end of the well so that the sample is
distributed across the well space. Avoid creating large air bubbles as you
dispense the mixture as they could lead to spillover.

Figure 5. Drag and dispense method – Start dispensing when the pipette tip is near the top of the well.
Finish dispensing when the pipette tip is near the bottom of the well.

2 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.
miRNA microarray slides processed with fewer than 8 samples can result in
gridding errors in Feature Extraction, which can require manual gridding.

Add the microarray slide

1 Remove a microarray slide from the slide storage box between your thumb
and index finger, numeric barcode side facing up and Agilent label facing down.
2 Use the four chamber base guideposts and rectangular end of the base to
position the microarray slide as you lower it to within 3 mm (1/8”) above the
gasket slide, making sure the microarray slide is not tilted with respect to the
gasket slide. Barcode ends of both the gasket slide and the microarray slide
must line up at the corners of the chamber base. Once positioned, gently rest
the microarray slide on the lower gasket slide.

32 miRNA Microarray System with miRNA Complete Labeling and Hyb Kit Protocol
2 Procedures
Step 3. Prepare the hybridization assembly

Refer to Figure 6 for proper technique on holding the microarray slide with
both hands. Although the image uses a 4-well gasket slide and 4-pack
microarray slide, the technique is the same for an 8-well gasket slide and
8-pack microarray slide.

Figure 6. Chamber base with gasket and microarray slide applied, guide posts denoted with arrows

Do not drop the microarray slide onto the gasket slide as this increases the
CAU TI O N chances of sample mixing between gasket wells.
Once placed, do not attempt to move the chamber and sandwiched slides as
this can cause leakage of the hybridization solution.

Assemble the chamber

1 Place the chamber cover, correct side facing up, onto the chamber base
which contains the “sandwiched” slides.

Correct Wrong
Figure 7. Chamber cover in correct (left) and incorrect (right) orientations

miRNA Microarray System with miRNA Complete Labeling and Hyb Kit Protocol 33
2 Procedures
Step 3. Prepare the hybridization assembly

2 From the rounded corner of the chamber base, slip the clamp onto the
chamber base and cover until it stops firmly in place, resting at the center of
the two pieces.
Keep the chamber assembly flat on the lab bench to avoid spilling the
hybridization solution.

Figure 8. Slipping the clamp onto the chamber base

3 Firmly tighten the thumbscrew fully.

The slides will not be harmed by hand-tightening.

Figure 9. Tightening of the thumbscrew on the clamp

If you do not completely tighten the thumbscrew, hybridization solution can

CAU TI O N leak out during hybridization.
Do not use tools to tighten the thumbscrew. The use of pliers or other tools can
damage the parts and will void the warranty.

4 Rotate the final assembled chamber in a vertical orientation, clockwise, 2 to 3

times to wet the gaskets (see Figure 10).
Rotation helps ensure that the hybridization solution will coat the entire
surface of the microarray during the incubation process.

34 miRNA Microarray System with miRNA Complete Labeling and Hyb Kit Protocol
2 Procedures
Step 3. Prepare the hybridization assembly

Figure 10. Rotation of the final assembled chamber

5 Inspect for good bubble formation.

• Hold the chamber vertically and inspect for stray or small bubbles that do
not move as you rotate the chamber.
• Use the “large mixing bubble” to dislodge small stray or stationary bubbles.
• If the small stray or stationary bubbles persist, gently tap the assembled
chamber on a firm surface. Rotate the chamber on its sides as you tap.
Inspect again and repeat if needed until the small stray or stationary
bubbles dissipate.

Wrong
Correct

Figure 11. The slide on the left shows a stray, stationary bubble (denoted with arrow), which must be
removed before hybridization. The slide on the right shows only large mixing bubbles, which move freely
around the chamber when rotated. Bubbles are acceptable, as long as they move freely when you rotate the
chamber.

miRNA Microarray System with miRNA Complete Labeling and Hyb Kit Protocol 35
2 Procedures
Step 4. Hybridize

Step 4. Hybridize

1 Load the assembled chamber into the oven rotator rack, starting from the
center of the rack (position 3 or 4 when counting from left to right). Refer to
the figure below for correct and incorrect orientations.

Figure 12. Assembled chambers in correct (left) and incorrect (middle and right) orientations

2 Close the door and set the rotator speed to 20 rpm.

3 Hybridize at 55°C for 20 hours.

Be sure that the arrays are hybridized for at least 20 hours. Hybridization can
CAU TI O N
occur for longer than 20 hours but the actual hybridization time should be
consistent if the results are to be compared. Failure to maintain consistent
hybridization times may adversely affect your data.

If you are not loading all the available positions on the hybridization rotator
CAU TI O N
rack, be 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
N OTE that you prepare the wash buffer the night before you plan to do the microarray
wash. See “Step 2. Prewarm Gene Expression Wash Buffer 2 and slide-staining
dish #3”.

36 miRNA Microarray System with miRNA Complete Labeling and Hyb Kit Protocol
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.

miRNA Microarray System with miRNA Complete Labeling and Hyb Kit Protocol 37
2 Procedures
Step 2. Prewarm Gene Expression Wash Buffer 2 and slide-staining dish #3

Step 2. Prewarm Gene Expression Wash Buffer 2 and

slide-staining dish #3

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.
3 Place slide-staining dish #3 (see Table 17 on page 40) for numbering of the
wash dishes) into a 1.5 L glass dish three-fourths filled with water. Warm to
37°C by storing overnight in an incubator set to 37°C.

Step 3. Prepare the equipment

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

Solvent wash
Wash staining dishes, racks and stir bars with isopropyl alcohol to avoid wash
artifacts on your slides and images.

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.

38 miRNA Microarray System with miRNA Complete Labeling and Hyb Kit Protocol
2 Procedures
Step 4. Wash the microarray slides

6 Discard the solvent as is appropriate for your site.

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
CAU TI O N
detergent in the washing of the staining dishes. If detergent is used, all traces
must be removed by copiously rinsing with Milli-Q water.

Step 4. Wash the microarray slides

The microarray wash procedure must be done in environments where ozone

N OTE 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
N OTE bath containing the pre-warmed Wash 2 solutions.

Table 17 lists the wash conditions for the wash procedure.

miRNA Microarray System with miRNA Complete Labeling and Hyb Kit Protocol 39
2 Procedures
Step 4. Wash the microarray slides

Table 17 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 5 minutes

2nd wash 3 Gene Expression Wash Buffer 2 37°C 5 minutes

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 prewarmed 1.5 L glass dish filled with water and containing
slide-staining dish #3 on a magnetic stir plate with heating element. Fill the
slide-staining dish #3 approximately three-fourths full with
Gene Expression Wash Buffer 2 (warmed to 37°C). Add a magnetic stir bar.
Turn on the heating element and maintain temperature of
Gene Expression Wash Buffer 2 at 37°C. Use a thermometer to check the
temperature.
4 Remove one hybridization chamber from incubator and record time. Record
whether bubbles formed during hybridization and if all bubbles are rotating
freely.
5 Prepare the hybridization chamber disassembly.
a Put the hybridization chamber assembly on a flat surface and loosen the
thumbscrew, turning counter-clockwise.

Figure 13. Loosening of the thumbscrew

b Slide off the clamp assembly and remove the chamber cover.

40 miRNA Microarray System with miRNA Complete Labeling and Hyb Kit Protocol
2 Procedures
Step 4. Wash the microarray slides

Figure 14. Removal of the clamp

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!

miRNA Microarray System with miRNA Complete Labeling and Hyb Kit Protocol 41
2 Procedures
Step 4. Wash the microarray slides

Figure 15. Removal of the microarray slide from the staining dish

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 5 minutes.
9 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 5 minutes.

You must maintain Gene Expression Wash Buffer 2 at 37°C for the duration of
CAU TI O N
the wash step. Failure to do so can result in alterations of stringency, which
can reduce the consistency of experimental results.

10 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.
11 Discard used Gene Expression Wash Buffer 1 and
Gene Expression Wash Buffer 2.
12 Repeat step 1 through step 11 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.

42 miRNA Microarray System with miRNA Complete Labeling and Hyb Kit Protocol
2 Procedures
Step 5. Put slides in a slide holder

Step 5. Put slides in a slide holder

For SureScan microarray scanner

1 Before you insert the slide into a slide holder, inspect the slide holder for any
dust or fingerprints. If found, remove the dust or fingerprints with compressed
air or a soft, dust-free cloth. If the slide holder is scratched, worn, or damaged,
has a lid that does not close tightly, or has a hinge that does not move freely,
discard the slide holder and select a different one.
2 Place the slide holder on a flat surface, with the clear cover facing up, and the
tab on the right. This helps to ensure that you have the slide aligned properly
when you insert it into the slide holder.
3 Gently push in and pull up on the tabbed end of the clear plastic cover to open
the slide holder.

Figure 16. Opening the slide holder

4 Insert the slide into the holder.

a Hold the slide at the barcode end and position the slide over the open slide
holder. Make sure that the active microarray surface faces up with the
barcode on the left, as shown in Figure 17.
b Carefully place the end of the slide without the barcode label onto the slide
ledge. See Figure 17.

miRNA Microarray System with miRNA Complete Labeling and Hyb Kit Protocol 43
2 Procedures
Step 5. Put slides in a slide holder

Slide ledge
Figure 17. Inserting slide into the slide holder

c Gently lower the slide into the slide holder. See Figure 18.
d Close the plastic slide cover, pushing on the tab end until you hear it “click.”
This moves the slide into position in the holder.

An improperly inserted slide can damage the scanner.

CAU TI O N

e Gently push in and pull up on the tabbed end of the clear plastic cover to
open it again and verify that the slide is correctly positioned.
f Close the plastic slide cover, gently pushing on the tab end until you hear it
“click”. See Figure 19. Make sure that the slide holder is completely closed.

Figure 18. Slide inserted in slide holder – cover open

44 miRNA Microarray System with miRNA Complete Labeling and Hyb Kit Protocol
2 Procedures
Step 5. Put slides in a slide holder

Figure 19. Slide inserted in slide holder – cover closed

For Agilent Scanner 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 20.
Refer to the Agilent Ozone-Barrier Slide Cover User Guide (publication
G2505-90550), included with the slide cover, for more information.

Figure 20. Inserting the ozone-barrier slide cover (shown for 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.
• 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.

miRNA Microarray System with miRNA Complete Labeling and Hyb Kit Protocol 45
2 Procedures
Scanning and Feature Extraction

Scanning and Feature Extraction

This section describes how to scan and extract data from miRNA microarrays.
Refer to the Agilent Microarray Scanner User Guide for more information on how
to use the scanner.
Table 18 Scanner to GeneSpring Workflow version compatibility

Microarray Scanner Scan Scan settings/profile Spike-In Feature Extraction GeneSpring

Format version Control version version
version

8x60K SureScan 9.X AgilentG3_miRNA Yes 10.7.3 or later 11.5 or later

8x60K SureScan 9.X AgilentG3_miRNA No 10.5 or later 10.0 or later

8x15K SureScan 9.X AgilentHD_miRNA Yes 10.7.3 or later 11.5 or later

8x15K SureScan 9.X AgilentHD_miRNA No 10.5 or later 10.0 or later

8x60K C 8.X AgilentG3_miRNA Yes 10.7.3 or later 11.0 or later

8x60K C 8.X AgilentG3_miRNA No 10.5 or later 10.0 or later

8x15K C 8.X AgilentHD_miRNA Yes 10.7.3 or later 11.0 or later

8x15K C 8.X AgilentHD_miRNA No 10.5 or later 10.0 or later

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]/cs/library/usermanuals/Public/G1662-90043_ScannerCom
[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.

46 miRNA Microarray System with miRNA Complete Labeling and Hyb Kit Protocol
2 Procedures
Step 1. Scan the slides

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. Refer to Figure 21 and
Figure 22.

Arrow helps identify

scanner orientation Finger-hold

Figure 21. Slide holder helps you to insert slides correctly

miRNA Microarray System with miRNA Complete Labeling and Hyb Kit Protocol 47
2 Procedures
Step 1. Scan the slides

Figure 22. Inserting slide holder into cassette

2 Select the appropriate scanner protocol:

• AgilentG3_miRNA (for G3 format)
• AgilentHD_miRNA (for HD format)
3 Verify that the Scanner status in the main window says Scanner Ready.
4 Click Start Scan.

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 AgilentG3_miRNA (for SurePrint G3 formats) or AgilentHD_miRNA (for
SurePrint HD formats).
4 Verify scan settings for -color scans. See Table 19.

Do not scan G3 microarrays with HD format settings. The resolution of the

CAU TI O N
resulting image will not be high enough for data analysis.

48 miRNA Microarray System with miRNA Complete Labeling and Hyb Kit Protocol
2 Procedures
Step 2. Extract data using Agilent Feature Extraction Software

Table 19 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.

Step 2. Extract data using Agilent Feature Extraction

Software

Feature Extraction (FE) is the process by which information from probe features
is extracted from microarray scan data, allowing researchers to measure miRNA
expression in their experiments. To get the most recent Feature Extraction
software for gene expression, go to the Agilent web site at
[Link]/[Link]?pageId=2058.
FE version 9.5.3 or higher is required for extraction of .tif images of Agilent
miRNA microarrays scanned on an Agilent Scanner. FE version 10.7 gives you
the option to automatically download new grid templates, protocols and QC
metrics (QCM or QCMT) via eArray, and supports analysis of the spike-ins
miRNA. See “Automatic Download from eArray” on page 54 for configuration.
Refer to Table 18 on page 46 to determine the version of Feature Extraction you
need to use.
FE version 10.7 protocol uses surrogates in determining the gTotalGeneSignal
which eliminates negative expression values. Earlier versions of FE and FE
protocols do not use surrogates and may create negative gTotalGeneSignal
values.

miRNA Microarray System with miRNA Complete Labeling and Hyb Kit Protocol 49
2 Procedures
Step 2. Extract data using Agilent Feature Extraction Software

After generating the microarray scan images, extract .tif images using the
Feature Extraction software.
For more information about normalizing one-color microarray data, see
“Normalizing Agilent One-Color Microarray Data” on page 58.

1 Open the Agilent Feature Extraction (FE) program.

To get the most recent Feature Extraction protocols for gene expression, go
to [Link]/[Link]?pageId=2058.
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 -Color miRNA FE protocol, the default miRNA
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 23 are selected.
d For versions of FE earlier than 10.7.3, in the Other section, from the QC
Metric Set drop-down list, select miRNA_QCMT_Jan09 as shown in
Figure 23 on page 51.
If the QC Metric Set is not available to select from the pull down menu, you
must add it to the QC Metric Set Browser. To add, right-click inside the QC
Metric Set Browser, and click Add. Browse for the QC Metric Set file and
click Open to load the QC Metric Set into the FE database.
If you don't have a local copy of the QC Metric Set or would like to
download the latest QC Metric Set, you can do so from Agilent Web site at
[Link]/[Link]?pageId=2058. After downloading,
you must add the QC Metric Set to the QC Metric Set Browser.

50 miRNA Microarray System with miRNA Complete Labeling and Hyb Kit Protocol
2 Procedures
Step 2. Extract data using Agilent Feature Extraction Software

After a new QC Metric Set is added to the QC Metric Set Browser,

remember to specify the default protocol for the new QC Metric Set if you
want the Feature Extraction program to automatically assign a FE QC
Metric Set to an extraction set.

Figure 23. Feature Extraction version 9.5 dialog box

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.

miRNA Microarray System with miRNA Complete Labeling and Hyb Kit Protocol 51
2 Procedures
Step 2. Extract data using Agilent Feature Extraction Software

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. For Agilent miRNA microarrays, select
miRNA_107_Sept09.
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]/[Link]?pageId=2058 to download the
latest protocols.

These FE Protocols were optimized using data from Agilent catalog arrays, which
N OTE have 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
N OTE version 9.5 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
N OTE from the B 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.

52 miRNA Microarray System with miRNA Complete Labeling and Hyb Kit Protocol
2 Procedures
Step 2. Extract data using Agilent Feature Extraction Software

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 25.
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.

Agilent Feature Extraction Result Files

Agilent Feature Extraction software generates two text files for each miRNA
microarray. The text file that uses the name
ScannerNum_BarcodeNum_S0#_FEProtocol_Array_Num.txt (for example,
“US22502705_251911810634_S01_miRNA-v1_95_May07_1_1.txt”) includes all
feature, probe and gene level data, as well as array level statistics and
parameters. Use these files for analysis and to review the QC metrics in the
GeneSpring GX version 10 or later.
The other text file, the “GeneView” file, uses the name
BarcodeNum_SO#_Array_Num_GeneView.txt (for example,
“251911810634_S01_1_1_GeneView.txt”). The GeneView file provides the
summarized TotalGeneSignal, TotalGeneError, and a detection flag for each
miRNA gene. This simple file is appropriate for any analyses and can be loaded
into GeneSpring GX version 9.0, or similar software for analysis. However, it does
not contain the QC metric data for review. The “gTotalGeneSignal” column has
been background subtracted and outlier rejected and is appropriate for use as
the measured intensity for each miRNA gene without further manipulation.
Please refer to the Agilent Feature Extraction Software User Guide or Reference
Guide for more detail on the data summarization algorithms and for information
on other columns in the feature extraction result files.

miRNA Microarray System with miRNA Complete Labeling and Hyb Kit Protocol 53
2 Procedures
Step 2. Extract data using Agilent Feature Extraction Software

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 24.

Figure 24. eArray Login Setting

54 miRNA Microarray System with miRNA Complete Labeling and Hyb Kit Protocol
2 Procedures
Step 2. Extract data using Agilent Feature Extraction Software

Figure 25. Example of a QC Report for an Agilent miRNA microarray, generated by Feature Extraction version 12.1 with the use of
Spike-In miRNAs.

miRNA Microarray System with miRNA Complete Labeling and Hyb Kit Protocol 55
2 Procedures
Step 2. Extract data using Agilent Feature Extraction Software

56 miRNA Microarray System with miRNA Complete Labeling and Hyb Kit Protocol
3 Supplemental Procedures
Normalizing Agilent One-Color Microarray Data 58
Characterization of Total RNA 60

The procedures in this chapter are supplemental to the main protocol.

miRNA Microarray System with miRNA Complete Labeling and Hyb Kit Protocol 57
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.
The appropriate normalization method depends on the sample type and
experimental design. Agilent recommends to use the signal value of the 90th
percentile of all of non-control probes on the microarray as a start. Adjust this
value as needed.

To do downstream analysis of Agilent microarray data

• Use GeneSpring GX 11.5 or later.

Note that the default normalization scheme for data in the GeneSpring GX 11.5
(or later) program is 90th percentile scaling.
For more information on the GeneSpring GX program, go to
[Link]/chem/genespring.

58 miRNA Microarray System with miRNA Complete Labeling and Hyb Kit Protocol
3 Supplemental Procedures
To use Feature Extraction

To use Feature Extraction

To normalize Agilent one-color microarray data without the GeneSpring program,

use the 90th 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 90th 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 90th
percentile signal (gPercentileIntensityProcessedSignal) to generate the 90th
percentile normalized microarray processed signals.
You can further scale the resulting 90th percentile-normalized signals by a
constant, such as the average of the 90th 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 Feature Extraction Software Reference Guide.

miRNA Microarray System with miRNA Complete Labeling and Hyb Kit Protocol 59
3 Supplemental Procedures
Characterization of Total RNA

Characterization of Total RNA

Although this step is optional in the miRNA microarray workflow, Agilent strongly
recommends that you characterize the total RNA for sample integrity and
possible presence of contaminants. This chapter gives a general guideline for
total RNA and small RNA characterization prior to proceeding with labeling and
hybridization.
Depending on the method used for purification of the total RNA, your sample
may have contaminants such as phenol, salts, and/or genomic DNA. You can
check the absorption spectrum from 220 nm to about 300 nm to detect some of
these possible contaminants. In the absorption spectra, you should see only one
peak with an absorption maximum at 260 nm. You may also see a shoulder of an
additional peak at < 220 nm that overlaps with and inflates the absorption at 260
nm. The level of inflation can be seen on the absorption spectrum. If the
absorption spectrum is not available, calculate the ratio of absorbances at
260/230 nm. This ratio should be greater than 1.8. If the ratio is less than 1.8,
then you need to purify the sample further by doing additional extractions with
chloroform, followed by an ethanol precipitation. To ensure recovery of the RNA,
do this purification with a large (microgram) quantity of total RNA.
Unfortunately, absorption spectra cannot differentiate between RNA and DNA.
Contamination with DNA can result in overestimation of total RNA amounts, and
may lead to decreased sensitivity on the miRNA assay due to the effective
decrease in the amount of total RNA added.
Characterization of the input total RNA using the Agilent 2100 bioanalyzer
provides information on the sample quality prior to labeling and hybridization.
You can use the RNA 6000 Nano kit to analyze total RNA with the appropriate
assay at the assay specified concentration. For low concentration samples, use
the RNA 6000 Pico kit.
The small RNA assay characterizes the total RNA sample with an emphasis on
the small RNA content of which a fraction is miRNA. This can be done using the
Small RNA Kit along with the Small RNA assay on the Agilent 2100 bioanalyzer.
For 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.

60 miRNA Microarray System with miRNA Complete Labeling and Hyb Kit Protocol
3 Supplemental Procedures
Step 1. Prepare for characterization of total RNA

For general assistance on the evaluation of total RNA with emphasis on the RNA
integrity number, refer to the application note “RNA integrity number (RIN) -
Standardization of RNA quality control,” part number 5989-1165EN.
To download application notes and for more information regarding Agilent’s
2100 bioanalyzer along with the various kits offered for analysis of both total
RNA and small RNA, visit the Web site at [Link]/chem/labonachip.

Step 1. Prepare for characterization of total RNA

• Refer to Table 20 and Table 21 to make sure that you have the appropriate
analyzer, kits, and compatible assays.
Table 20 Analyzer and Kits

Description Company and part no.

2100 Bioanalyzer Agilent p/n G2938C or G2939A

RNA 6000 Nano Kit Agilent p/n 5067-1511

RNA 6000 Pico Kit Agilent p/n 5067-1513

Small RNA Kit Agilent p/n 5067-1548

Table 21 Recommended Assays

Description Compatible Assay

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

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

Small RNA Kit Agilent Small RNA Assay

• 1 to 100 ng/μL of total RNA
• 100 to 2000 pg miRNA
• 1 to 20 ng/μL of purified small RNA < 200 nt

miRNA Microarray System with miRNA Complete Labeling and Hyb Kit Protocol 61
3 Supplemental Procedures
Step 2. Characterize the RNA samples using the Agilent 2100 Bioanalyzer

Step 2. Characterize the RNA samples using the Agilent

2100 Bioanalyzer

To measure the total RNA integrity, purity and concentration, use one of the
Agilent RNA 6000 kits. Follow the instructions in the kit.
To measure the small RNA amount and profile, use the Agilent Small RNA kit.
Follow the instructions in the kit.

1 Ensure the 2100 bioanalyzer electrodes have been cleaned as instructed in

the reagent kit guide.
2 Open the Agilent 2100 expert software (version B.02.04 or higher), switch on
the 2100 bioanalyzer and check communication.
3 Prepare the chip, samples and ladder as instructed in the reagent kit guide.
4 Load the prepared chip into the 2100 bioanalyzer and start the run within five
minutes after preparation.
5 Within the instrument context, choose the appropriate assay from the drop
down list.
6 Start the run. Enter sample names and comments in the Data and Assay
context.
7 Verify the results.

RNA 6000 - Total RNA

Total RNA characterization provides information on the quality of the total RNA,
which may impact the overall RNA quantification and the microarray miRNA
profile.
The resulting electropherogram typically has at least two distinct peaks
representing the 18S and 28S ribosomal RNA. The peak representing the lower
marker can also be seen as shown in Figure 26. The presence of the 5S RNA
peak, as shown in Figure 26, depends on the purification method, and generally
shows lower abundance in column purified total RNA. Figure 26 demonstrates
the differences in the peaks with varying degrees of total RNA integrity and
provides RNA Integrity Number (RINs) as examples. Make sure you define a
minimum threshold RIN number based on correlative data in order to eliminate
experimental bias due to poor total RNA quality.

62 miRNA Microarray System with miRNA Complete Labeling and Hyb Kit Protocol
3 Supplemental Procedures
Step 2. Characterize the RNA samples using the Agilent 2100 Bioanalyzer

18S
28S

5S

LM

Figure 26. 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.

Small RNA - miRNA in total RNA

The small RNA assay characterizes the total RNA sample with an emphasis on
the small RNA content of which a fraction is miRNA.
The resulting electropherogram typically has a number of bands or peaks
representing small RNAs ranging in size from 10 to 150 nt. The miRNA portion is
represented by the band or peak ranging in size from 10 to 40 nt. These bands or
peaks will vary in abundance depending on the total miRNA preparation.
Figure 27 shows an example of the resulting electropherogram of the small RNA
assay for a total RNA sample.
You can record the results of the small RNA assay to correlate to your miRNA
microarray data. However, the small RNA assay results may not necessarily be
an indicator for the miRNA content in the sample, and the miRNA microarray
assay is a more sensitive technique for detecting low levels of miRNAs.

miRNA Microarray System with miRNA Complete Labeling and Hyb Kit Protocol 63
3 Supplemental Procedures
Step 2. Characterize the RNA samples using the Agilent 2100 Bioanalyzer

Figure 27. Characterization of small RNA within a total RNA sample. miRNA is typically found in the 10 to 40 nt region, which is a
subset of the small RNA that range from 10 to 150 nt

64 miRNA Microarray System with miRNA Complete Labeling and Hyb Kit Protocol
4 Reference
Kit Contents 66
Supplemental User Guides 67
“Secure Fit” Slide Box Opening Instructions 68
Microarray Handling Tips 70
General Microarray Layout and Orientation 71
Array/Sample tracking microarray slides 74

This chapter contains reference information related to the protocol and Feature
Extraction default parameter settings

miRNA Microarray System with miRNA Complete Labeling and Hyb Kit Protocol 65
4 Reference
Kit Contents

Kit Contents

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

microRNA Spike-In Kit

Dilution Buffer
Labeling Spike-In
Hyb Spike-In

miRNA Complete Labeling and Hyb Kit

10× Gene Expression Blocking Agent
2× Hi-RPM Hybridization Buffer
T4 RNA Ligase
10× T4 RNA Ligase Buffer
Calf Intestinal Phosphatase
10× Calf Intestinal Phosphatase Buffer
DMSO
nuclease-free water
Cyanine 3-pCp

Gene Expression Wash Buffer Kit

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

66 miRNA Microarray System with miRNA Complete Labeling and Hyb Kit Protocol
4 Reference
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 platform workflow.
• 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

miRNA Microarray System with miRNA Complete Labeling and Hyb Kit Protocol 67
4 Reference
“Secure Fit” Slide Box Opening Instructions

“Secure Fit” Slide Box Opening Instructions

Agilent now ships all microarray slides in a newly designed “secure fit” slide box.
The instructions below describe how to remove the slide box from the shipping
pouch, how to open the slide box, and how to properly remove a microarray slide.
1 Use scissors to cut below the seal and remove box from its foil pouch.
After breaking foil on microarray pouch, store microarray slides in the slide
box at room temperature (in the dark) under a vacuum desiccator or nitrogen
purge box.

Figure 28. Opening foil pouch (left) and removing the slide box (right)

2 Place the slide box on a flat surface. While stabilizing the box from the top
with one hand, use a sharp edge to cut the sealing tape on both sides of the
box before opening.

Figure 29. Cutting the sealing tape

68 miRNA Microarray System with miRNA Complete Labeling and Hyb Kit Protocol
4 Reference
“Secure Fit” Slide Box Opening Instructions

3 With one hand, firmly hold the base of the box on the sides with the
indentations (or dimples) for added grip.

Figure 30. Gripping the base at the indentations (top) and close-up of the indentations (bottom)

4 Using your free hand, grasp the lid and gently lift it away from the base as if it
is hinged. Set the lid aside.
Depending on your preference, you can reverse the hand positions so that the
left hand holds the base while the right hand grasps the lid.

Figure 31. Grasping the lid (left) and lifting the lid from the base (right)

miRNA Microarray System with miRNA Complete Labeling and Hyb Kit Protocol 69
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
CAU TI O N
for use 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.

70 miRNA Microarray System with miRNA Complete Labeling and Hyb Kit Protocol
4 Reference
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 32. 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 32 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.

miRNA Microarray System with miRNA Complete Labeling and Hyb Kit Protocol 71
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 33).
This changes the feature numbering and location as it relates to the “microarray
design files” For more information, go to
[Link]/cs/library/technicaloverviews/public/G4502-90002_G3Micr
[Link] 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.

72 miRNA Microarray System with miRNA Complete Labeling and Hyb Kit Protocol
4 Reference
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 33. Microarray slide orientation

miRNA Microarray System with miRNA Complete Labeling and Hyb Kit Protocol 73
4 Reference
Array/Sample tracking microarray slides

Array/Sample tracking microarray slides

Use the form 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.
L

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

Barcode Number __________________________________________________________

Figure 34. 8-pack microarray slide

74 miRNA Microarray System with miRNA Complete Labeling and Hyb Kit Protocol
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75 miRNA Microarray System with miRNA Complete Labeling and Hyb Kit Protocol
In This Book
This document describes the Agilent
recommended procedures for the
preparation and labeling of complex
biological targets and hybridization, washing,
scanning, and feature extraction of Agilent
oligonucleotide microarrays for
microarray-based miRNA systems.

[Link]
Agilent Technologies, Inc. 2010- 2015, 2021
October 2021
Revision C1

*G4170-90011*
G4170-90011 Version 4.1