Expression profiling using affymetrix genechip probe arrays

Abstract

Large-scale microarray expression profiling studies have helped us to understand basic biological processes and to classify and predict the prognosis of cancers; they have also accelerated the identification of new drug targets. Affymetrix GeneChip probe arrays are high-density oligonucleotide microarrays that are available for many prokaryotic and eukaryotic species. Affymetrix human and mouse whole-genome microarrays analyze the expression level of up to 47,000 transcripts and variants. Each transcript is measured by 11 probe pairs, which consist of a perfect match 25mer oligonucleotide (PM) and a 25mer mismatch oligonucleotide (MM) that contains a single base pair mismatch in the central position. The PM/MM design is used for identification and subtraction of nonspecific hybridization and background signals. Advantages of Affymetrix GeneChip arrays include highly standardized array fabrication, as well as standardized target preparation, hybridization, and processing protocols, resulting in low technical variability and good reproducibility between experiments. This chapter describes the standard assay procedures for isolating total RNA from heart tissue, generating a biotin-labeled target for expression analysis, processing of Affymetrix GeneChip probe arrays using the Affymetrix instrument system, and quality control measures.