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Genomic and RNA Profiling Core Facility

Houston, Texas

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Genomic and RNA Profiling Core
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Affymetrix Expression Arrays

Affymetrix offers four different whole genome arrays for expression analysis. They include the 3' IVT array, the exon array, the gene array and the whole transciptome array. The figure below illustrates the probe coverage for each array type.

Affymetrix Expression Array Probe Distribution-Courtesy of Affymetrix.com


3'IVT Plus (replaces the 3'IVT Express kit)

The Affymetrix 3'IVT Plus kit uses the same basis for generating biotin-labeled cRNA as the 3'IVT Express Kit, which can then be hybridized to 3' targeting probes on Affymetrix's GeneChip expression arrays. Affymetrix has optimized the kit to generate cRNA with slightly longer lengths, which increases confidence in data integrity since the 3'IVT arrays have probe sets which are, by design, closer to the 3'-end transcripts. In general, comparison between the two kits shows very similar cRNA yield amounts, average technical reproducibility, % Median Coefficient of Variation (%CV), % Probes present (which indicates similar detection sensitivity), and very high correlation (Pearson r = 0.975 - 0.992). Please see this link for an in depth review of the 3'IVT Express and 3'IVT Plus comparison. Also available is the 3'IVT Plus Reagent Kit Data Sheet.

Please see the table below for starting sample inputs and available GeneChip arrays.

3’ IVT Express (Discontinued after March 15, 2014)

The Affymetrix 3' IVT Express kit is used for visualization of 3' biased eukaryotic expression. This platform utilizes oligo-dT primers to produce cDNA, then in vitro transcription to generate biotin-labeled cRNA, which is hybridized to 3' targeting probes on the array. It was designed to replace the one- and two-cycle expression kits previously available from Affymetrix. While using the same basic chemistry as its predecessors, it has been optimized to allow for lower sample input and faster processing times, while retaining comparable result quality and reproducibility. Affymetrix GeneChip Arrays

3' IVT Express Kit
IVT Incubation Time GARP Recommended Minimum total RNA input GARP Recommended Minimum Concentration
4 hours
500 ng (250 ng)*
167 ng/µL (84 ng/µL)*
16 hours
250 ng (50 ng)*
84 ng/µL (17 ng/µL)*
*Minimum Assay Requirements


Prokaryotic Expression

The Affymetrix Prokaryotic Assay measures RNA expression levels in prokaryotic samples. It employs random hexamer primers to produce cDNA, which is fragmented, labeled with terminal transferase and biotin, and hybridized onto a Prokaryotic array. Affymetrix GeneChip Arrays

Prokaryotic Expression
Total RNA input Minimum Concentration
10 µg
556 ng/µL


Whole Transcript

The Whole Transcript assay allows researchers to accurately look at overall gene expression by covering the entire length of the gene and not just the 3' end. Exon arrays enable users to detect alternative splicing events of a particular gene transcript or measure whole transcript expression. Gene arrays, on the other hand, offer whole-genome, whole-transcript expression analysis for human, mouse, or rat genomes. Affymetrix GeneChip Arrays

Whole Transcript Assays
Array Type Total RNA input Minimum Concentration
Gene Arrays
100 ng
34 ng/µL
Exon Arrays
100 ng
34 ng/µL


Whole Transcriptome

Go beyond gene-level expression profiling with Affymetrix's next generation GeneChip® Human Whole Transcriptome Array 2.0. This assay provide increased coverage required to accurately detect transcript isoforms within a family of transcripts. This high-resolution array contains an unprecedented >6.0 million probes covering coding transcripts and non-coding transcripts. 70% of the probes on this array cover exons for coding transcripts, and the remaining 30% of probes on the array cover exon-exon splice junctions and non-coding transcripts. The unparalleled coverage of this array provides the deepest insight into all coding and non-coding transcripts available. With Affymetrix' Expression Console (version 1.3) and their newly developed Transcriptome Analysis Console (TAC) softwares, researchers can translate and summarize data into gene-level, exon level and splice-junction probe sets. This array is currently offered for the human transcriptome. Find more information about this array and software. For a list of available Affymetrix GeneChip Arrays.

Human Whole Transcriptome Assay
Total RNA input Minimum Concentration
100 ng
34 ng/µL

For users wanting to submit FFPE RNA samples to be used with Affymetrix' Whole Transcriptome platform, GARP recommends using the two following RNA Isolation protocols before submitting FFPE RNA samples for SAQC. Qiagen FFPE Recoverall Protocol and Qiagen FFPE RNeasy Protocol.


miRNA (utilizing Genisphere FlashTag™ Biotin Labeling Assay)

Affymetrix currently offers two GeneChip® miRNA arrays, versions 2.0 and their new miRNA 3.0 arrays.

The GeneChip® miRNA 2.0 array is a single, comprehensive microRNA (miRNA) array with probes covering 71 organisms, including human, mouse, rat, and monkey. The probe contents of the array are derived from Sanger’s miRBase miRNA database V11 in addition to over 900 human snoRNAs and scaRNAs. Samples are labeled with the Genisphere FlashTag™ Biotin Labeling Assay, which utilizes the 3DNA™ technology. The 3DNA™ dendrimer is ligated to samples to allow multiple biotins (~15) to bind to each poly-A tailed, RNA molecule. Following FlashTag™ ligation, samples are hybridized on the Affymetrix GeneChip® miRNA 2.0 array overnight. Affymetrix GeneChip Arrays

miRNA 2.0 (utilizing Genisphere FlashTagTM Biotin Labeling Assay)
RNA Input Type Genisphere Recommended Minimum total RNA input Minimum Concentration
Total RNA
1 µg (0.1 µg)*
125 ng/µL (20 ng/uL)*
Enriched LMW RNA from Total RNA
Enriched from
1 µg (0.1 µg)* total RNA
Enriched from
125 ng/µL (20 ng/uL)* total RNA
*Minimum Assay Requirements

Affymetrix's new GeneChip® miRNA 3.0 array is a single, comprehensive microRNA (miRNA) array with probes covering 153 organisms, including human, mouse, rat, and monkey. The probe contents of the array are derived from Sanger’s miRBase miRNA database V17 in addition to over 2,216 human snoRNAs and scaRNAs. Samples are labeled with the Genisphere FlashTag™ Biotin HSR RNA Labeling Assay, which utilizes the 3DNA™ technology. The 3DNA™ dendrimer is ligated to samples to allow multiple biotins (~15) to bind to each poly-A tailed, RNA molecule. Following FlashTag™ ligation, samples are hybridized on the Affymetrix GeneChip® miRNA 3.0 array overnight. Affymetrix GeneChip Arrays

miRNA 3.0 (utilizing Genisphere FlashTagTM Biotin HSR RNA Labeling Assay)
RNA Input Type
Genisphere Recommended Minimum total RNA input
Minimum Concentration
Total RNA
1 µg (0.1 µg)*
130 ng/µL (20 ng/uL)*
Enriched LMW RNA from Total RNA
Enriched from
1 µg (0.1 µg)* total RNA
125 ng/µL (20 ng/uL)* total RNA
*Minimum Assay Requirements

NuGEN for Affymetrix

NuGEN has revolutionized amplification and labeling technologies that enable life science researchers to mine genomic samples from extremely small, degraded, and hard-to-replace specimens. The Ovation® RNA Amplification System V2 uses as little as 5 ng of total RNA (although at least 20 ng is recommended). This amplification system is 3’ bias, but only one round of amplification is performed which covers ~1500 bases from the poly A tail.

For samples with even stricter concentration restraints, the Ovation™ Pico WTA System V2 requires only 500 pg of total RNA and a single round of amplification. This system incorporates oligo dT and random primers for amplification at the 3’ end and throughout the whole transcriptome. Consequently, partially degraded RNA samples prepared using the WT-Ovation™ Pico System provide adequate amplified product.

For severely degraded samples, such as FFPE tissue derived RNA samples, NuGEN provides the Ovation™ FFPE WTA System. This system also amplifies throughout the entire transcriptome in addition to the 3’ end. Intact total RNA has been tested on this system with successful results, requiring only small amount of sample input.

Amplified RNA samples from these systems are subsequently hybridized to Affymetrix GeneChip® arrays overnight. Please reference the chart below to determine NuGEN system and Affymetrix array compatibility. For more information about NuGEN Product, please click here.

NuGEN Ovation FFPE Workflow

Affymetrix Expression Alternative Labeling – NuGEN Technologies, Inc.

System Compatible Affymetrix Arrays Minimum Total RNA Input Minimum Concentration
Ovation RNA Amplification System V2
3' Expression
50 ng
10 ng/µL
Ovation Pico WTA System V2

3' Expression

WT-Exon

WT- Gene

500 pg
100 pg/µL
Ovation FFPE WTA System

3' Expression

WT-Exon

WT- Gene

10 ng (intact RNA)

50 ng (degraded RNA)

2 ng/µL (intact)

10 ng/µL (degraded)

For users wanting to submit FFPE samples, GARP recommends using NuGEN's Prelude FFPE RNA Isolation kit, which is included with the Ovation FFPE WTA System, or an isolation kit specially designed for FFPE tissues manufactured by Qiagen or Zymo. For customers wanting to submit FFPE samples for microarray analysis using NuGEN's Ovation FFPE WTA System, please contact GARP to request the kit order and submit billing information. Please include a note that you will want to use the Prelude FFPE RNA Isolation kit enclosed with WTA kit. Since GARP does not perform RNA or DNA isolations, users will need to pick up the Prelude kit and perform these isolations prior to submitting RNA samples for SAQC. Users will be billed for the entire Ovation FFPE WTA kit, no partial billing.


Ambion for Affymetrix

Ambion’s MessageAmp™ II-Biotin Enhanced Single Round aRNA Amplification Kit uses ArrayScript™ reverse transcriptase to produce high yields of first strand cDNA, ensuring that even low abundance mRNAs are reverse transcribed into full length cDNAs. Optimal concentrations of biotinylated nucleotides are incorporated into samples during amplification and labeling. For lower amounts of total RNA input, two rounds of amplification are required. The first round of amplification requires the MessageAmp™ II aRNA Amplification Kit to produce unmodified aRNA, which is carried through to the MessageAmp™ II-Biotin Enhanced Single Round aRNA Amplification Kit for a second round of IVT.

Amplified RNA samples from these kits are subsequently fragmented on Affymetrix’s platform and hybridized to Affymetrix GeneChip® arrays overnight.

Affymetrix Expression Alternative Labeling – Ambion
Kit Type Recommended Total RNA Input Recommended Minimum Concentration

MessageAmp™ II-Biotin Enhanced Single Round aRNA Amplification Kit

1 µg (50 ng)*
100 ng/µL (5 ng/uL)*
Double Amplification: Message Amp™ aRNA and MessageAmp™ II-Biotin Enhanced Single Round aRNA Amplification Kits
100 ng (100 pg)*
10 ng/µL (10 pg/uL)*
*Minimum Assay Requirements

Please visit the Affymetrix website for array formats and organism types.

NOTE: All samples must pass GARP Sample Quality Control (SAQC) before they can be submitted for array processing. More GARP Sample Submission Specifications for Affymetrix and Agilent Microarrays.

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