| Microarray Analysis
Affymetrix
3420 Central Expressway
Santa Clara, CA 95051 USA
E-mail: support@affymetrix.com
408-731-5000
www.affymetrix.com/genechip/expression
Affymetrix Whole-transcript Sense Target Labeling Assay Protocol
The Affymetrix Whole-transcript Sense Target Labeling Assay is
Whole-transcript Labeling 3’ IVT Assay
**
Assay
*
(Starting Material: 1-15 µg total RNA
designed to generate unbiased, amplifi ed and biotinylated sense-
(Starting Material: 100 ng or 1 µg total RNA) or 0.2-2 µg mRNA)
strand DNA targets from the entire expressed genome. Using the
0 h
Day 1
0 h
Poly-A RNA Control Addition Poly-A RNA Control Addition
Day 1 Kit: Poly-A RNA Control Kit: Poly-A RNA Control
Whole-transcript Assay in combination with GeneChip
®
Exon 1.0 ST
and Gene 1.0 ST Arrays allows you to measure expression across the 1.5 h RNA Reduction and RNA cDNA Synthesis 3.5 h
Concentration
†
Kit: One-cycle cDNA Synthesis
entire length of a gene, thus offering a more complete and accurate
Kit: RiboMinus
™
Transcriptome Isolation
picture of overall gene expression than 3´-based assays.
4.5 h cDNA Synthesis Cleanup of Double-stranded cDNA 0.5 h
Kit: WT cDNA Synthesis and Kit: Sample Cleanup Module
Overnight
Amplification
This protocol employs a combination of random-priming, linear
In vitro Transcription Biotin Labeling of Antisense cRNA
Kit: WT cDNA Synthesis and Kit: IVT Labeling
Amplification Overnight
amplifi cation and a novel, robust fragmentation and labeling strategy
Day 2
0.5 h Cleanup of Antisense RNA (cRNA) Cleanup of Biotinylated cRNA 0.5 h
to generate targets along the entire length of a transcript. As little as
Day 2 Kit: Sample Cleanup Module Kit: Sample Cleanup Module
100 ng of total RNA starting material is required for the assay. 2.5 h cDNA Synthesis and cRNA Hydrolysis Fragmentation 1 h
Kit: WT cDNA Synthesis and Kit: Sample Cleanup Module
Amplification
As shown in Figure 1, the protocol begins with the addition of Poly-A
0.5 h Cleanup of Sense Strand DNA Hybridization/Washing/Staining
Kit: Sample Cleanup Module Kit: Hybridization, Wash, and Stain
RNA Controls to total RNA and is optionally followed by a ribosomal
Overnight
3 h Fragmentation and Labeling
RNA reduction step when using the RiboMinus
TM
version of this
Kit: WT Terminal Labeling
protocol. From here, cDNA synthesis and in vitro transcription are
Overnight
Hybridization/Washing/Staining
performed, followed by a cleanup of the antisense RNA. The purifi ed
Kit: Hybridization, Wash, and Stain
antisense RNA is then used for another round of cDNA synthesis.
Day 3
10-35 min
†† Scanning Scanning
<12 min
After fragmentation and labeling, the sense-strand cDNA target is
Day 3
~2.5 days ~2.5 days
subsequently hybridized to either Exon or Gene 1.0 ST Arrays.
*Whole-transcript (WT) Sense Target Labeling Assay
**GeneChip® One-Cycle Target Labeling Assay
†
Affymetrix has optimized this assay to produce the most sensitive
The RNA reduction step is omitted in the 100ng Total RNA Labeling Protocol
††
10 minutes for gene arrays and 35 minutes for exon arrays
and specifi c expression data when used in conjunction with Exon
and Gene Arrays. Overall workfl ow time from sample RNA to array
Figure 1: Comparison of whole-transcript assay (100 ng and 1 µg,
data (~2.5 days) remains similar to previous 3´-biased IVT assays.
RiboMinus
TM
protocols) versus the 3´ IVT assay.
References:
Okoniewski M., et al. High Correspondence Between Affymetrix Exon and
Standard Expression Arrays. BioTechniques 42(2):181-185 (2007).
French P. J., et al. Identification of Differentially Regulated Splice Variants
and Novel Exons in Glial Brain Tumors Using Exon Expression Arrays. Cancer
Research 67:5635-5642 (2007).
Protocol Guide ı 2008 View entire protocol online at www.biotechniques.com/protocol ı BioTechniques ı 47
Affymetrix Protocol.indd 47 10/26/07 11:44:26 AM
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