Thermo Fisher Scientific GeneChip User Manual
GeneChip™ 3' IVT PLUS Reagent Kit
USER GUIDE
Manual Target Preparation for GeneChip™ 3' Expression Arrays
Catalog Numbers 902415 and 902416
Publication Number MAN0018104
Revision B.0
For Research Use Only. Not for use in diagnostic procedures.
Thermo Fisher Scientific Baltics
UAB |
V.A. Graiciuno 8, LT-02241 |
Vilnius, Lithuania
Aymetrix Pte Ltd |
7 Gul Circle #2M-01 |
Keppel Logistics Building |
Singapore 629563
Products:
GeneChip™ 3' IVT PLUS Reagent Kit
Products:
GeneChip™ 3′ Expression Arrays
For descriptions of symbols on product labels or product documents, go to thermofisher.com/symbols-definition.
The information in this guide is subject to change without notice.
DISCLAIMER: TO THE EXTENT ALLOWED BY LAW, THERMO FISHER SCIENTIFIC INC. AND/OR ITS AFFILIATE(S) WILL NOT BE
LIABLE FOR SPECIAL, INCIDENTAL, INDIRECT, PUNITIVE, MULTIPLE, OR CONSEQUENTIAL DAMAGES IN CONNECTION WITH OR
ARISING FROM THIS DOCUMENT, INCLUDING YOUR USE OF IT.
Revision history: Pub. No. MAN0018104
Revision Date Description
B.0 28 October 2020 Changed volume to load on array for the 169/400 format to 90 μL.
Updated image of GeneChip™ cartridge array.
A.0 27 December 2018 Initial release in Thermo Fisher Scientific document control system.
Supersedes legacy Aymetrix™ publication number 703210.
Updated to the current document template, with associated updates to trademarks, logos,
licensing, and warranty.
Important Licensing Information: These products may be covered by one or more Limited Use Label Licenses. By use of these
products, you accept the terms and conditions of all applicable Limited Use Label Licenses.
TRADEMARKS: All trademarks are the property of Thermo Fisher Scientific and its subsidiaries unless otherwise specified. Agencourt
and SPRI are trademarks of Agencourt Bioscience. Agilent and Bioanalyzer are trademarks of Agilent Technologies, Inc. Corning is a
trademark of Corning, Inc. LabChip is a trademark of Caliper Life Sciences, Inc., part of PerkinElmer, Inc. Oligo is a trademark of Oligo
LLC Limited Liability Company Delaware. Tough-Spots is a trademark of Diversified Biotech, Incorporated Corporation.
©2020 Thermo Fisher Scientific Inc. All rights reserved.
Contents
■
CHAPTER 1 The GeneChip™ 3' IVT PLUS Reagent Kit .......................... 5
Purpose ........................................................................ 5
Assay workflow ................................................................. 6
Kit contents and storage ......................................................... 7
Required materials .............................................................. 8
Instruments ................................................................ 8
Reagents and supplies ...................................................... 9
■
CHAPTER 2 Protocol .............................................................. 11
Procedural notes ............................................................... 11
Implement a plan to maintain procedural consistency .......................... 11
Program the thermal cycler ................................................. 12
How to handle kit components .............................................. 13
Prepare Control RNA ........................................................... 13
Prepare Control RNA ....................................................... 13
Prepare poly-A RNA controls ................................................ 14
Prepare total RNA .............................................................. 16
Evaluate RNA quality ....................................................... 16
Evaluate RNA integrity ...................................................... 16
Determine RNA quantity .................................................... 17
Prepare total RNA/poly-A RNA control mixture ................................ 17
Synthesize first-strand cDNA .................................................... 18
Synthesize second-strand cDNA ................................................. 19
Synthesize labeled cRNA by in vitro transcription .................................. 20
Purify labeled cRNA ............................................................ 21
Assess cRNA yield and size distribution ........................................... 23
Expected cRNA yield ....................................................... 23
Determine cRNA yield by UV absorbance ..................................... 25
(Optional) Expected cRNA size distribution ................................... 25
(Optional) Expected cRNA size distribution using a Bioanalyzer™ Instrument ...... 26
Fragment labeled cRNA ......................................................... 27
GeneChip™ 3' IVT PLUS Reagent Kit Assay Manual Workflow User Guide
3
Contents
■
■
CHAPTER 3 3′ array hybridization ............................................... 29
Cartridge array hybridization on the GeneChip™ Instrument ......................... 29
Prepare ovens, arrays, and sample registration files ............................ 29
Target hybridization setup for cartridge arrays ................................. 29
Wash and stain the cartridge arrays .......................................... 33
Scan the cartridge arrays ................................................... 33
Array strip hybridization on the GeneAtlas™ Instrument ............................. 34
Target hybridization setup for array strips ..................................... 34
GeneAtlas™ software setup ................................................. 39
Rehybridizing used cocktails ................................................ 45
Array plate hybridization on the GeneTitan™ MC Instrument ......................... 46
Target hybridization setup for array plates .................................... 46
Hybridization setup ........................................................ 48
Process 3' array plates on the GeneTitan™ MC Instrument ...................... 49
APPENDIX A cRNA purification photos ......................................... 51
■
APPENDIX B Safety ............................................................... 53
Chemical safety ................................................................ 54
Biological hazard safety ......................................................... 56
■
Documentation and support ....................................................... 57
Related documentation ......................................................... 57
Customer and technical support ................................................. 57
Limited product warranty ........................................................ 57
4
GeneChip™ 3' IVT PLUS Reagent Kit Assay Manual Workflow User Guide
1
Purpose .............................................................................. 5
■
Assay workflow ....................................................................... 6
■
Kit contents and storage ............................................................... 7
■
Required materials ..................................................................... 8
■
Purpose
The GeneChip™ 3' IVT PLUS Reagent Kit enables you to prepare RNA samples for gene-expression
profiling analysis with GeneChip™ 3′ Expression Arrays. The kit generates amplified and biotinylated
complementary RNA (cRNA) from poly(A) RNA in a total RNA sample. cRNA is also known as amplified
RNA or aRNA. The kit does not need an up-front removal of ribosomal RNA and is optimized for use
with GeneChip™ 3′ Expression Arrays.
The GeneChip™ 3' IVT PLUS
Reagent Kit
The GeneChip™ 3' IVT PLUS Reagent Kit uses a reverse-transcription priming method that primes the
poly(A) tail junction of RNA to provide gene-expression profiles from mRNA. RNA amplification is based
on linear amplification and employs T7 in vitro transcription (IVT) technology. The kit is comprised of
reagents and a protocol for preparing hybridization-ready targets from 50 to 500 ng of total RNA. See
“Assay workflow” on page 6.
The 3′ IVT PLUS reagent is optimized to work with total RNA from a wide range of samples including
tissues, cells, and cell lines. Total RNA from whole blood samples should be processed for globin
reduction prior to target preparation with the GeneChip™ 3' IVT PLUS Reagent Kit.
GeneChip™ 3' IVT PLUS Reagent Kit Assay Manual Workflow User Guide
5
Chapter 1 The GeneChip™ 3' IVT PL
1
Assay workflow
Assay workflow
US Reagent Kit
Figure 1 The 3′ IVT PL
6
US amplification and labeling process.
GeneChip™ 3' IVT PLUS Reagent Kit Assay Manual Workflow User Guide
Chapter 1 The GeneChip™ 3' IVT PLUS Reagent Kit
Kit contents and storage
Table 1 GeneChip™ 3' IVT PLUS Reagent Kit contents and storage.
Kit contents and storage
1
Amount,
Component
3′ IVT PLUS Amplification Kit Module 1
3′ First-Strand Enzyme 11 µL 50 µL
3′ First-Strand Buer 500 µL 500 µL
3′ Second-Strand Enzyme 22 µL 70 µL
3′ Second-Strand Buer 55 µL 180 µL
3′ IVT Enzyme 66 µL 210 µL
3′ IVT Buer 500 µL 1,580 µL
3′ IVT Biotin Label 44 µL 140 µL
Control RNA (1 mg/mL
HeLa total RNA)
Nuclease-free Water 1 x 1.0 mL 2 x 1.0 mL From –20°C to room
3′ IVT PLUS Amplification Kit Module 2
10-reaction kit
(902415)
5 µL 5 µL
Amount,
30-reaction kit
(902416)
Storage
–20°C
temperature
3′ Fragmentation Buer 1 mL 1 mL Room temperature
Purification Beads 1.1 mL 3.3 mL 4°C (Do not freeze.)
GeneChip™ Eukaryotic Poly-A RNA Control Kit (900433)
Poly-A Control Stock 16 µL 16 µL
–20°C
Poly-A Control Dil Buer 3.8 mL 3.8 mL
GeneChip™ Hybridization Contr
20X Hybridization Controls 450 µL 450 µL
3 nM Control Oligo™ B2 150 µL 150 µL
Tubes Organizer: Plastic vinyl template for organization and storage of components in 9 x 9 array, 81-places square
wells, 5.25 x 5.25 x 2 in. (133 x 133 x 52 mm) (for example, Nalgene™ Polycarbonate 9 x 9 CryoBox™ 5026-0909 , or
equivalent).
ol Kit (900454)
–20°C
GeneChip™ 3' IVT PLUS Reagent Kit Assay Manual Workflow User Guide
7
Chapter 1 The GeneChip™ 3' IVT PLUS Reagent Kit
1
Required materials
Required materials
Instruments
Unless otherwise indicated, all materials are available through thermofisher.com. "MLS" indicates that
the material is available from fisherscientific.com or another major laboratory supplier.
Table 2 Instruments required for target preparation
•
Agencourt™ SPRI™ Plate Super Magnet Plate or
•
Magnetic Stand-96 or
•
Magnetic-Ring Stand (96 well)
•
or equivalent
Item Source
•
A32782 or
•
AM10027 or
•
AM10050
•
or equivalent
Microcentrifuge
NanoDrop™ UV-Vix Spectrophotometer Or equivalent quantitation
(Optional) Agilent™ 2100 Bioanalyzer™ Instrument or equivalent DNA and
RNA sizing instrument
Pipettes
Thermal cycler
Vortex mixer
65°C heat block or oven for incubation of Nuclease-free Water during
purification
MLS
instrument
MLS
MLS
MLS
MLS
MLS
Table 3 Instruments required for array processing
Instruments Source
GeneChip™ System 3000Dx v.2 for cartridge arrays (Cat. No. 00-0334)
GeneChip™ Hybridization Oven 645 00-0331 (110/220V)
GeneChip™ Fluidics Station 450 00-0079
GeneChip™ Scanner 3000 7G System 00-0213
GeneChip™ AutoLoader with External Barcode Reader 00-0090 (GCS 3000 7G S/N 501)
00-0129 (GCS 3000 7G S/N 502)
GeneAtlas™ System for array strips (Cat. Nos. 00-0394, 00-0375)
GeneAtlas™ Workstation 90-0894
8
GeneChip™ 3' IVT PLUS Reagent Kit Assay Manual Workflow User Guide
Chapter 1 The GeneChip™ 3' IVT PLUS Reagent Kit
Required materials
Table 3 Instruments required for array processing (continued)
Instruments Source
GeneAtlas™ Hybridization Station 00-0380 (115VAC)
00-0381 (230VAC)
GeneAtlas™ Fluidics Station 00-0377
GeneAtlas™ Imaging Station 00-0376
GeneAtlas™ Barcode Scanner 74-0015
GeneTitan™ system for array plates
1
GeneTitan™ Multi-Channel (MC) Instrument, NA/Japan includes 110v
UPS
GeneTitan™ Multi-Channel (MC) Instrument, Int′l includes 220v UPS 00-0373
GeneTitan™ Multi-Channel (MC) Instrument, NA/Japan includes 110v
UPS
GeneTitan™ Multi-Channel (MC) Instrument, Int′l Includes 220v UPS 00-0363
Reagents and supplies
Unless otherwise indicated, all materials are available through thermofisher.com. "MLS" indicates that
the material is available from fisherscientific.com or another major laboratory supplier.
Table 4 Additional reagents and supplies required
Corning™ Clear Polystyrene 96-Well Microplate Fisher Scientific™, 07-200-103
GeneChip™ Hybridization, Wash, and Stain Kit (30 reactions) 900720
GeneAtlas™ Hybridization, Wash, and Stain Kit for 3' IVT Arrays
(60 reactions)
00-0372
00-0360
Item Source
901531
GeneTitan™ Hybridization, Wash, and Stain Kit for 3' IVT Arrays
(96 reactions)
100% Ethanol (molecular biology grade or equivalent)
Nuclease-free Water for preparing 80% ethanol wash solution
Nuclease-free aerosol-barrier tips
Nuclease-free 1.5, and 0.2 mL tubes or plates
Nuclease-free 15 mL tubes or containers
Amber 1.5 mL tubes (for processing cartridge arrays only)
(Optional) 96-well plate sealing film
GeneChip™ 3' IVT PLUS Reagent Kit Assay Manual Workflow User Guide
901530
MLS
MLS
MLS
MLS
MLS
MLS
MLS
9
Chapter 1 The GeneChip™ 3' IVT PLUS Reagent Kit
1
Required materials
Table 4 Additional reagents and supplies required (continued)
Item Source
(Optional) Reagent reservoir for multichannel pipette
(Optional) Agilent™ RNA 6000 Nano Kit or equivalent DNA and RNA
sizing reagents
Tough-Spots™ labels
MLS
50671511
MLS
10
GeneChip™ 3' IVT PLUS Reagent Kit Assay Manual Workflow User Guide
2
Procedural notes ..................................................................... 11
■
Prepare Control RNA .................................................................. 13
■
Prepare total RNA .................................................................... 16
■
Synthesize first-strand cDNA ........................................................... 18
■
Synthesize second-strand cDNA ....................................................... 19
■
Synthesize labeled cRNA by in vitro transcription ......................................... 20
■
Purify labeled cRNA .................................................................. 21
■
Assess cRNA yield and size distribution ................................................. 23
■
Fragment labeled cRNA ............................................................... 27
■
Procedural notes
Protocol
Implement a plan to maintain procedural consistency
Subtle procedural dierences in gene-expression assays can cause sample-to-sample variation. To
minimize this variation, implement a detailed procedural plan that standardizes the variables in the
procedure. The plan should address the following topics.
•
Method of RNA isolation
•
Amount of input RNA used for each tissue type
•
RNA purity and integrity
•
Equipment preparation
•
Reagent preparation
•
Workflow stopping points
GeneChip™ 3' IVT PLUS Reagent Kit Assay Manual Workflow User Guide
11
2
Chapter 2 P
Procedural notes
rotocol
Program the thermal cycler
Set the temperature for the heated lid to or near the required temperature for each step. An alternate
protocol may be used for thermal cyclers that lack a programmable heated lid. These protocols are
described in Table 5. However, this is not the preferred method.
Yields of cRNA may be greatly reduced if a heated lid is used during the second-strand cDNA
synthesis or during the in vitro transcription cRNA synthesis steps. Leave the heated lid open during
second-strand cDNA synthesis. A small amount of condensation will form during the incubation. This is
expected, and should not significantly decrease cRNA yields. For in vitro transcription cRNA synthesis,
incubate the reaction in a 40°C hybridization oven if a programmable heated-lid thermal cycler is
unavailable.
Incubation temperatures and times are critical for eective RNA amplification. Use properly calibrated
thermal cyclers and adhere closely to the incubation times.
Ensure that the heated lid of your thermal cycler tracks the temperature of the thermal cycling block or
supports specific temperature programming.
IMPORTANT! Concentr
ation fluctuations caused by condensation can aect yield. Ensure that the
heated-lid feature of the thermal cycler is working properly.
Table 5 Thermal cycler protocols.
Protocol
First-Strand cDNA
Synthesis
Second-Strand cDNA
Synthesis
In Vitro T
cRNA Synthesis
Fragmentation 94°C 105°C 94°C for
Hybridization Control 65°C 105°C 65°C for
Hybridization Cocktail 99°C 105°C 95°C or 99°C
[1]
[2]
ranscription
F
or thermal cyclers that lack a programmable heated lid.
Four hours for 250—500 ng RNA input, or 16 hours for 50—250 ng RNA input.
Heated lid
t
emperature
42°C 105°C 42°C for
Room
temperature
or disable
40°C 40°C oven 40°C for
Alternate
protocol
Lid open 16°C for
[1]
Step 1 Step 2 Step 3 Volume
2 hours
1 hour
4 hours or
16 hours
35 minutes
5 minutes
for 5 minutes
[2]
4°C for
2 minutes
65°C for
10 minutes
4°C, hold
4°C, hold
45°C for
5 minutes
4°C for
2 minutes
10 µL
30 µL
60 µL
Varies
Varies
Varies
12
GeneChip™ 3' IVT PLUS Reagent Kit Assay Manual Workflow User Guide
How to handle kit components
Chapter 2 Protocol
Prepare Control RNA
2
IMPORTANT! Reagents in the GeneChip
maximum of 3 times.
•
Enzymes: Mix by gently vor
the tube, then keep on ice.
•
Buers: Thaw on ice, thoroughly vortex to dissolve precipitates, then centrifuge briefly to collect
contents at the bottom of the tube. If necessary, warm buers at ≤37°C for 1–2 minutes or until the
precipitate is fully dissolved, then keep on ice.
•
Purification beads: Allow to equilibrate at room temperature before use.
•
Prepare master mixes for each step of the procedure to save time, improve reproducibility, and
minimize pipetting error.
•
Prepare master mixes as follows:
–
Prepare only the amount needed for all samples in the experiment plus ~5% overage to
account for pipetting loss.
–
Use nonstick nuclease-free tubes to prepare the master mix.
–
Add enzymes last and just before adding the master mix to the reaction.
•
Return all components to the recommended storage temperature immediately after use.
Prepare Control RNA
™
3' IVT PLUS Reagent Kit can be thawed and frozen a
texing the tube, centrifuge briefly to collect contents at the bottom of
Prepare Control RNA
To verify that the reagents are working as expected, a Control RNA (1 mg/mL HeLa total RNA) sample is
included with the kit. Use this procedure to prepare the Control RNA for a positive control reaction.
1.
On ice, dispense 2 µL of the Control RNA in 78 µL of Nuclease-free Water, for a total volume of
80 µL (25 ng/µL).
2.
Follow the procedure in “Prepare total RNA/poly-A RNA control mixture” on page 17, but use 2 µL
of the diluted Control RNA (50 ng) in the control reaction.
Note:
Measure the concentration of HeLa Control RNA with a NanoDrop™ UV-Vix Spectrophotometer.
·
Use the measured concentration for calculation and for preparing the 25 ng/µL working stock.
The positive control reaction should produce >15 µg of cRNA from 50 ng of Control RNA, using
·
a 16-hour incubation for the IVT reaction.
GeneChip™ 3' IVT PLUS Reagent Kit Assay Manual Workflow User Guide
13
Chapter 2 Protocol
2
Prepare Control RNA
Prepare poly-A RNA controls
Note:
To include premixed controls from the GeneChip™ Eukaryotic Poly-A RNA Control Kit, add the
·
reagents to the total RNA samples. Follow the procedure described in “Prepare total RNA/poly-A
RNA control mixture” on page 17. We strongly recommend the use of poly-A RNA controls for all
reactions that will be hybridized to GeneChip™ arrays.
If the Poly-A Control Dil Buer is frozen, allow 15–20 minutes to thaw at room temperature.
·
A supplied set of poly-A RNA controls provides exogenous positive controls to monitor the entire target
preparation. The control should be added to the RNA prior to the First-Strand cDNA Synthesis step.
Each eukaryotic GeneChip™ probe array contains probe sets for several B. subtilis genes that are
absent in eukaryotic samples (lys, phe, thr, and dap). These poly-A RNA controls are in vitro
synthesized, and the polyadenylated transcripts for the B. subtilis genes are premixed at staggered
concentrations. The concentrated Poly-A Control Stock can be diluted with the Poly-A Control Dil Buer
and spiked directly into RNA samples to achieve the final concentrations, referred to as a ratio of copy
number, summarized in Table 6.
Table 6 Final concentrations of poly-A RNA controls when added to total RNA samples.
Poly-A RNA spike Final concentration (ratio of copy number)
lys 1:100,000
phe 1:50,000
thr 1:25,000
dap 1:6,667
The controls are then amplified and labeled together with the total RNA samples. The hybridization
intensities of these controls on GeneChip™ arrays help the operator monitor the labeling process
independently from the quality of the starting RNA samples.
The Poly-A Control Stock and Poly-A Control Dil Buer are provided in the GeneChip™ Eukaryotic
Poly-A RNA Control Kit to prepare the appropriate serial dilutions based on Table 7. This is a guideline
when 50, 100, 250, or 500 ng of total RNA is used as starting material. For starting sample amounts
other than those listed here, calculate the appropriate dilutions to arrive at the same proportionate final
concentration of the spike-in controls in the samples.
14
GeneChip™ 3' IVT PLUS Reagent Kit Assay Manual Workflow User Guide
Chapter 2 P
Prepare Control RNA
Table 7 Serial dilutions of poly-A RNA control stock.
Serial dilutions Volume of
Total RNA input
amount
50 ng 1:20 1:50 1:50 1:20 2 µL
100 ng 1:20 1:50 1:50 1:10 2 µL
250 ng 1:20 1:50 1:50 1:4 2 µL
500 ng 1:20 1:50 1:50 1:2 2 µL
First dilution Second dilution Third dilution Fourth dilution
rotocol
fourth dilution
to add to total
RNA
IMPORTANT!
void pipetting less than 2 µL of any solution to maintain precision and consistency when preparing
A
·
the dilutions.
Use nonstick nuclease-free tubes to prepare all of the dilutions. Nonstick tubes are not included in
·
the kit.
After each step, mix the poly-A control dilutions thoroughly. Vortex them gently, then centrifuge them
·
quickly to collect contents at the bottoms of the tubes.
2
For example, to prepare the poly-A RNA dilutions for 100 ng of total RNA:
1.
Add 2 µL of the P
dilution (1:20).
oly-A Control Stock to 38 µL of the Poly-A Control Dil Buer to prepare the first
Tip: The first dilution of the poly-A RNA controls can be stored for up to 6 weeks in a non-frost-
free freezer at −20°C. It can be frozen and thawed up to 8 times. Label the storage tube with its
expiration date.
2.
Add 2 µL of the first dilution t
(1:50).
3.
Add 2 µL of the second dilution to 98 µL of Poly-A Control Dil Buer to prepare the third dilution
(1:50).
4.
Add 2 µL of the third dilution to 18 µL of Poly-A Control Dil Buer to prepare the fourth dilution
(1:10).
5.
Add 2 µL of this fourth dilution to 100 ng of total RNA. The final volume of total RNA with the
diluted poly-A controls should not exceed 5 µL.
o 98 µL of Poly-A Control Dil Buer to prepare the second dilution
GeneChip™ 3' IVT PLUS Reagent Kit Assay Manual Workflow User Guide
15
Chapter 2 Protocol
2
Prepare total RNA
Prepare total RNA
Evaluate RNA quality
Total RNA samples should be free of genomic DNA and we recommend including a DNase treatment or
genomic DNA removal step with the RNA purification method. The contaminating genomic DNA may be
amplified along with the RNA, which will lead to inaccurate measurement of transcriptome expression.
In addition, the contaminating genomic DNA could cause over-estimation of the RNA amount.
RNA quality aects how eciently an RNA sample is amplified using this kit. High-quality RNA is free
of contaminating proteins, DNA, phenol, ethanol, and salts. To evaluate RNA quality, determine its
A
260/A280
Evaluate RNA integrity
The integrity of the RNA sample, or the proportion that is full length, is an important component of
RNA quality. Reverse transcribing partially-degraded mRNA may generate cDNA that lacks parts of the
coding region.
Methods to evaluate RNA integrity include the following:
•
•
ratio. RNA of acceptable quality is in the range of 1.7 to 2.1.
Microfluidic analysis, using the Agilent™ 2100 Bioanalyzer™ Instrument or equivalent instrument
with an RNA LabChip™ Kit or equivalent kit.
Denaturing agarose gel electrophoresis.
With microfluidic analysis, use the RNA Integrity Number (RIN) to evaluate RNA integrity. For more
information on how to calculate RIN, see www.genomics.agilent.com.
Denaturing agarose gel electrophoresis and nucleic acid staining separate and make visible the 28S
and 18S rRNA bands. The mRNA is likely to be full length if the 2 bands have these characteristics:
•
The 28S and 18S rRNA bands resolve into 2 discrete bands with no significant smearing below
each band.
•
28S rRNA band intensity is approximately twice that of the 18S rRNA band.
Note: Total RNA samples with lower RIN values may require increased input amounts to generate
enough labeled cRNA for hybridization to an array.
16
GeneChip™ 3' IVT PLUS Reagent Kit Assay Manual Workflow User Guide
Determine RNA quantity
Consider the type and the amount of sample RNA available when planning your experiment. Because
mRNA content varies significantly with tissue type, determine the total RNA input empirically for each
tissue type or experimental condition. The recommended total RNA inputs in Table 8 are based on total
RNA from HeLa cells. Use these values as reference points for determining the optimal RNA input.
Note: Avoid pipetting less than 2 µL of any solution to maintain precision and consistency. Predilute
high-concentration RNA samples with Nuclease-free Water before adding RNA to the first-strand cDNA
synthesis reaction.
Table 8 Input RNA limits.
RNA input Total RNA
Recommended 100 ng
Minimum 50 ng
Maximum 500 ng
Chapter 2 Protocol
Prepare total RNA
2
Table 9 Recommended IVT incubation time.
Recommendation RNA amount IVT incubation time
Recommended 50—250 ng 16 hours
Optional 250—500 ng 4 hours
Prepare total RNA/poly-A RNA control mixture
Prepare total RNA according to your laboratory’s procedure. A maximum of 5 µL total RNA can be
added to the first-strand synthesis reaction. If you are adding Poly-A Control Stock to your RNA, the
volume of the RNA must be 3 µL or less (Table 10). See “Prepare poly-A RNA controls” on page 14
for more information. For example, when performing the Control RNA reaction, combine 2 µL of RNA
(25 ng/µL), 2 µL of diluted Poly-A Control Stock, and 1 µL of Nuclease-free Water.
Note: When adding Poly-A Control Stock to RNA, the volume of RNA must be 3 µL or less. If
necessary, use a SpeedVac™ Vacuum Concentrator or ethanol precipitation to concentrate the RNA
samples.
Table 10 Total RNA/poly-A RNA control mixture.
Component Volume for 1 reaction
Total RNA sample (50—500 ng) Variable
Diluted Poly-A Control Stock (fourth dilution) 2 µL
Nuclease-free Water Variable
Total volume 5 µL
GeneChip™ 3' IVT PLUS Reagent Kit Assay Manual Workflow User Guide
17
Chapter 2 P
2
Synthesize first-strand cDNA
rotocol
Synthesize first-strand cDNA
In this reverse-transcription procedure, total RNA is primed with T7 oligo(dT) primer. The reaction
synthesizes single-stranded cDNA with the T7 promoter sequence at the 5′ end.
Note: Avoid pipetting less than 2 µL of any solution to maintain precision and consistency. Predilute
high-concentration RNA samples with Nuclease-free Water before adding the RNA to the first-strand
cDNA synthesis reaction.
1.
Prepare the First-Strand Master Mix.
a.
On ice, prepare the First-Strand Master Mix in a nuclease-free tube. Combine the components
in the sequence shown in the following table. Prepare the master mix for all the total RNA
samples in the experiment. Include ~5% overage for pipetting losses.
Table 11 First-Strand Master Mix.
Component Volume for 1 reaction
3′ First-Strand Buer 4 µL
3′ First-Strand Enzyme 1 µL
Total volume 5 µL
b.
Mix thor
of the tube. Proceed immediately to the next step.
c.
On ice, transfer 5 µL of the First-Strand Master Mix to each tube or well.
2.
Add the total RNA to each First-Strand Master Mix aliquot.
a.
On ice, add 5 µL of the total RNA from Table 10 to each (5 µL) tube or well containing the
First-Strand Master Mix, for a final reaction volume of 10 µL.
See “Prepare total RNA/poly-A RNA control mixture” on page 17.
b.
Mix thoroughly by gently vortexing the tube. Centrifuge briefly to collect the reaction at the
bottom of the tube or well, then proceed immediately to the next step.
3.
Incubate for 2 hours at 42°C, then for at least 2 minutes at 4°C.
a.
Incubate the first-strand synthesis reaction in a thermal cycler using the First-Strand cDNA
Synthesis protocol that is shown in Table 5.
b.
Immediately after the incubation, centrifuge briefly to collect the first-strand cDNA at the
bottom of the tube or well.
oughly by gently vortexing the tube. Centrifuge briefly to collect the mix at the bottom
18
GeneChip™ 3' IVT PLUS Reagent Kit Assay Manual Workflow User Guide
Loading...