Fluorescence intensities were normalized to the average fluorescence of the entire microarray. The detailed protocol for further processing of the arrays is available from Affymetrix.
After washing and staining, the fluorescence intensity was measured twice for each array using the GeneArray scanner (Affymetrix).
The generated biotin-labeled cRNA was fragmented by metal-induced hydrolysis, and was hybridized to the GeneChip array (HG-U133A, Affymetrix). The detailed protocol for hybridization, washing and staining of the arrays is available from Affymetrix. Microarray gene expression analysis was performed in triplicates as previously described (Oswald et al., 2006).
High-quality RNA was isolated with the RNeasy Mini Kit (Qiagen, Hilden, Germany) according to the manufacturers instructions.
5 ?g of total RNA were used per patient sample for first strand cDNA synthesis, according to the Eukaryotic Target Labeling protocol as recommended by the manufacturer. Synthesis of biotin-marked cRNA was performed as described in the in vitro-transcription protocol (Affymetrix, Santa Clara, CA, USA).