Published in
November
2007
(p.47)
DOI: 10.2144/000112647
The Affymetrix Whole-transcript Sense Target Labeling Assay is designed to generate unbiased, amplified and biotinylated sense-strand DNA targets from the entire expressed genome. Using the Whole-transcript Assay in combination with GeneChip® Exon 1.0 ST and Gene 1.0 ST Arrays allows you to measure expression across the entire length of a gene, thus offering a more complete and accurate picture of overall gene expression than 3'-based assays.
This protocol employs a combination of random-priming, linear amplification and a novel, robust fragmentation and labeling strategy to generate targets along the entire length of a transcript. As little as 100 ng of total RNA starting material is required for the assay.
As shown in Figure 1, the protocol begins with the addition of Poly-A RNA Controls to total RNA and is optionally followed by a ribosomal RNA reduction step when using the RiboMinus™ version of this protocol. From here, cDNA synthesis and in vitro transcription are performed, followed by a cleanup of the antisense RNA. The purified antisense RNA is then used for another round of cDNA synthesis. After fragmentation and labeling, the sense-strand cDNA target is subsequently hybridized to either Exon or Gene 1.0 ST Arrays.
Figure 1. (Click to enlarge)
Affymetrix has optimized this assay to produce the most sensitive and specific expression data when used in conjunction with Exon and Gene Arrays. Overall workflow time from sample RNA to array data (∼2.5 days) remains similar to previous 3'-biased IVT assays.
