Total RNA from whole tumors specimens was extracted by using the perfect RNA Eukaryotic Kit (Eppendorf, Hamburg, Germany). Instead , total RNA from LCM-derived material, was extracted by following the PicoPureTM RNA isolation kit manufacture’s instruction (Arcturus Engineering, Mountain View, CA). Total RNA from whole tumors specimens was extracted by using the perfect RNA Eukaryotic Kit (Eppendorf, Hamburg, Germany). Instead , total RNA from LCM-derived material, was extracted by following the PicoPureTM RNA isolation kit manufacture’s instruction (Arcturus Engineering, Mountain View, CA)., total RNA
Label
biotin
Label protocol
We applied the “small sample target labeling protocol” to amplify and to label targets from total RNA samples for GeneChip® probe array expression analysis (www.affymetrix.com).
Hybridization protocol
In order to identify unscheduled hybridations, each sample was tested with a test chip containing eukaryotic control sequences including housekeeping genes. Then each sample was probed with the Human Genome U133A GeneChip® (Affymetrix, Inc., CA, USA). After prehybridization, both single test and standard probe arrays were hybridized with biotinylated cRNA hybridization cocktail at 45°C for 16 hours in the Affymetrix GeneChip® Hybridization Oven 640. Probe array wash and stain were performed in the Fluidics station. Each chip was stained with a streptavidin-phycoerythrin conjugate.
Scan protocol
GeneChips were scanned using the laser confocal fluorescence scanner.
Description
Gene expression data from whole tumor
Data processing
Image acquisition and image analysis were performed using the Affymetrix® Microarray Suite Software The probe level data from the Human Genome 133A GeneChip® were pre-processed using the affy software package (Gautier et al 2003). No background correction was performed, and probe level intensities were normalized using the vsn (Huber et al. 2002) normalization method.