Extraction of total RNA from frozen tumors was carried out using the Qiagen RNeasy Mini Kit (Qiagen, Germany). The integrity of the RNA extracts was tested on an Agilent 2100 Bioanalyzer (Agilent Technologies, Rockville, MD), measuring the 28S: 18S ribosomal RNA ratio. RNA extracts of high quality were stored at -70C until microarray analyses.
Label
biotin
Label protocol
Preparation of in vitro transcription (IVT) products and oligonucleotide array hybridization and scanning were performed according to the Affymetrix protocol (Santa Clara, CA, USA). In brief, the amount of starting total RNA for each probe preparation varied between 2 and 5 μg. IVT reactions were performed in batches to generate biotinylated cRNA targets, which were subsequently chemically fragmented at 95C for 35 min.
Hybridization protocol
Fragmented and biotinylated cRNA (10 μg) was hybridized at 45C for 16 h to a high density oligonucleotide custom made Affymetrix array chip (Human Cancer G110 Array). The arrays were then washed, stained with streptavidin-phycoerythrin (SAPE, final concentration of 10 μg/ml).
Scan protocol
The arrays were scanned according to the manufacturer’s instructions (Affymetrix Genechip® Technical Manual, 2001). The scanned images were inspected for the presence of obvious defects (artifacts or scratches) on the array. In case of visible microarray artifacts, the sample was rehybridized and rescanned on new chips using the same fragmented probe.
Data processing
Probe intensities were extracted, background corrected, normalized and summarized to probeset expression using the rma function in the R/Bioconductor package affy with the default settings
