chapter  10
Affymetrix GeneChip analyses – the impact of RNA quality
ByLudger Klein-Hitpass, Tarik Möröy
Pages 14

According to the steeply increasing number of reports in the literature Affymetrix DNA oligonucleotide arrays (GeneChips) have gained considerable acceptance in the research community. The latest version of human GeneChips, U133A Plus 2.0, representing approximately 38 500 transcripts on a single chip allows genome-wide expression profiling in a very convenient setting. In contrast to cDNA and oligonucleotide arrays from different manufacturers, Affymetrix GeneChips measure transcripts by a set of multiple probes (called a probe set), which usually consists of 11 probe pairs. Each probe pair contains two 25mer oligonucleotide probes, a perfect match (PM) oligonucleotide that represents part of the cDNA sequence of interest and a mismatch (MM) oligonucleotide, which is identical to the PM oligo except for a mismatch mutation at the central position. In the Affymetrix image analysis method implemented in the MAS5.0 and GCOS software, the PM-MM signal differences of the 11 probe pairs of a probe set are converted into a single probe set signal value, which is a measure of the abundance of the transcript. In addition, for each probe set the software estimates the reliability of the measurement resulting in a detection call (present, absent, or marginal) based on a significance analysis of the PMMM differences.