Affymetrix Conventional GeneChips (e.g. U133 Plus 2.0 Arrays)
These arrays contain 11 25mer oligonucleotides (probes) that are a perfect match for each gene on the array. For this series of arrays, probe sets are clustered together in a ~300 bp region within 1 Kb of the 3í end of the transcript and frequently within 500 bp of the end. In addition, a corresponding set of oligos are present that contain a single base mismatch to the predicted sequence. The mismatched oligos (MM) serve as a control for potential cross hybridization. The current generation of whole 3í biased GeneChips detect 40-55,000 transcripts and include well annotated genes as well as ESTs and less annotated putative transcripts. To search the arrays for your gene of interest or to download complete gene lists for the Affymetrix arrays, please visit NetAffx*. For additional information see:
affymetrix.com*
Sample Preparation
Total RNA samples are isolated using either TRIzol or one of many commercial RNA isolation kits such as Qiagen RNAeasy. We require that the quality and quantity of each sample is checked using Agilent BioAnalyzer and Nanodrop spectrophotometer. Next, double stranded cDNA is prepared using an oligo(dT)-T7 RNA polymerase primer. Biotinylated cRNA is prepared by in vitro transcription and the resulting RNA is fragmented by partial hydrolysis in the presence of Mg++ and high temperature. The fragmented cRNA is then hybridized to the GeneChip, stained with phycoerythrin-streptavidin and imaged by standard procedures.
We typically use Ambion MessageAmp II- biotin enhanced kits for sample preparation which are cost effective and require a minimum of 250 ng total RNA in our hands. For smaller amounts of starting material (currently 500 pg total RNA minimum) see Small Sample Preparation.
Array Comparisons
*Links To Other Web Sites. Links to other web sites are provided solely as a convenience to you. If you use these links, you will be leaving the Gladstone Institutes website. Please note that The Gladstone Institutes is not responsible for any of these other websites or their content.
