The Affymetrix® protocol (Affymetrix, Santa Clara, CA) has been previously described [89]. Briefly, starting from 300ng of total RNA, cDNA synthesis and cRNA labeling were performed using the GeneChip® 3' IVT Express Kit (Affymetrix). Ten μg of fragmented cRNA were hybridized on the Affymetrix GeneChip® Human Genome U133A 2.0 (HG-U133A 2.0). This array provides comprehensive coverage of the transcribed human genome using 22,214 probesets, and captures the expression of ≈18,400 human transcripts. Each array was scanned on the Affymetrix GeneChip® Scanner 3000 7G (Affymetrix), and raw probe intensities stored in.CEL files by the GeneChip® Operating Software (GCOS v1.4). Array quality was assessed by monitoring the 3′/5′ ratios of GAPDH, and the percentage of “Present” genes (%P) and array exhibiting GAPDH 3′/5′ < 3.0 and %P > 40% were considered of good quality. Based on these metrics no arrays were excluded.
