Affymetrix Expression Arrays

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

Affymetrix Expression Array Probe Distribution-Courtesy of Affymetrix.com

3’ IVT Express

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.

**3' IVT Express Kit**
IVT Incubation Time	MCF Recommended Minimum total RNA input	MCF Recommended Minimum Concentration
4 hours	250 ng (100 ng)*	84 ng/µL (34 ng/µL)*
16 hours	100 ng (50 ng)*	34 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.

**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.

**Whole Transcript Assays**
Array Type	Total RNA input	Minimum Concentration
Gene Arrays	100 ng	34 ng/µL
Exon Arrays	1 µg	313 ng/µL

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.

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

**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 (0.1µg)*	130 ng/µL (20 ng/uL)*
Enriched LMW RNA from Total RNA	Enriched from 1 µg (0.1µg)* 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 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 WT-Ovation™ Pico System 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 WT-Ovation™ FFPE 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.

**Affymetrix Expression Alternative Labeling – NuGEN Technologies, Inc**.
System	Compatible Affymetrix Arrays	Minimum Total RNA Input	Minimum Concentration
Ovation RNA Amplification System	3' Expression	5 ng	1 ng/µL
WT-Ovation Pico System	3' Expression WT-Exon WT- Gene	500 pg	100 pg/µL
WT-Ovation FFPE System	3' Expression WT-Exon WT- Gene	10 ng (intact RNA) 50 ng (degraded RNA)	2 ng/µL (intact) 10 ng/µL (degraded)

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.

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