Total RNA was extracted from frozen tissue with a polytron homogenizer (Brinkman, Westbury, NY) and Trizol reagent (Invitrogen, Rockville, MD) and purified with an RNAse kit (Qiagen, Santa Clara, CA).
Label
Biotin
Label protocol
Total RNA was used as a template for double-stranded cDNA synthesis (Superscript Double-Stranded cDNA synthesis kit, Invitrogen, Carlsbad, CA). Biotin-labelled cRNA was synthesized by using Enzo Bioarray High Yield RNA transcription Labeling Kit (Affymetrix).
Hybridization protocol
Hybridized to Affymetrix Human Genome U133 Plus 2.0 arrays (Santa Clara, CA) according to the manufacturer’s instructions.
Scan protocol
The intensity of bound dye was measured with an argon laser confocal scanner (GeneArray Scanner, Agilent).
Data processing
The probe arrays were scanned twice and the stored images were analyzed using the Expression Console (Affymetrix, Santa Clara, CA). RMA data was derived by using Bioconductor.
