Total RNA was extracted from fresh tissues using RNeasy mini kit (Qiagen) following the manufacturer’s protocol using rotor-stator homogenization. Approximately 30 mg of fresh tissues was used for each sample. For extraction of RNA from paraffin-embedded tissue, the Optimum FFPE RNA isolation kit (Ambion) was used, using materials derived from four 8m sections. The non-tumor areas on the slides were manually removed with surgical blades, and the Total RNA was extracted from fresh tissues using RNeasy mini kit (Qiagen) following the manufacturer’s protocol using rotor-stator homogenization. Approximately 30 mg of fresh tissues was used for each sample. For extraction of RNA from paraffin-embedded tissue, the Optimum FFPE RNA isolation kit (Ambion) was used, using materials derived from four 8m sections. The non-tumor areas on the slides were manually removed with surgical blades, and the Total RNA was extracted from fresh tissues using RNeasy mini kit (Qiagen) following the manufacturer’s protocol using rotor-stator homogenization. Approximately 30 mg of fresh tissues was used for each sample. For extraction of RNA from paraffin-embedded tissue, the Optimum FFPE RNA isolation kit (Ambion) was used, using materials derived from four 8m sections. The non-tumor areas on the slides were manually removed with surgical blades, and the remaining tissue on the slide was scraped into an Eppendorf tube for RNA extraction.
Label
biotin
Label protocol
RNA was reverse-transcribed and in vitro transcribed and biotin-labeled using Affymetrix one-cycle cDNA synthesis and IVT labeling kits (Affymetrix)
Hybridization protocol
Following biotinylation and fragmentation, hybridization was performed against the Affymetrix Human HG-U133 plus 2.0 GeneChips according to the manufacturers directions.
Scan protocol
GeneChips were scanned using the Affymetrix GeneChip Scanner 3000.
Description
none
Data processing
Raw data probeset intensity data was extracted from the CEL files using the RMA algorithm in GeneSpring 7.2 and the data was then median normalized per chip.
