Total RNA was isolated from tumor samples using a TRIzol reagent (Invitrogen Life Technologies, Carlsbad, CA) and was followed by a cleanup on a RNeasy column (Qiagen, Hilden, Germany).
cRNA probes were generated using standard Affymetrix protocols.
Aliquots of each sample were hybridized to U133A oligonucleotide microarray using standard Affymetrix protocols.
The chips were scanned using the GeneArray scanner (Affymetrix) using standard Affymetrix protocols.
All RNA samples had a 28S/18S ratio over 1.5 with no evidence of degradation. All scanned images were visually inspected for surface defects. Quality Control assessment of array data was performed using GCOS v1.4 (Affymetrix, Santa Clara, Ca) and Array Assist v5.0 build 30237 (Stratagene, La Jolla, Ca). Hybridization and Poly-A controls were within expected parameters, and 3'/5' ratios for actin and GAPDH were below 3 (mean Beta Actin 3'/5' = 1.53 and mean GAPDH 3'/5' = 1.02). GCOS v1.4 Expression Reports also revealed expected Call percentages (Present: 48% to 62%; Absent: 36% to 49%; Marginal: 1.5% to 1.9%). All arrays were within 1.5 fold of each other in overall intensity.
The CEL files generated by the Affymetrix Microarray Suite (MAS 5.0) were converted into DCP files using the DNA-Chip Analyzer (dCHIP 1.3).