Frozen tumour samples were homogenized in Trizol Reagent (Invitrogen, Carlsbad, Canada) with a rotor-stator homogenizer, and RNA was extracted following the manufacturer's instructions. Trizol extracted RNA was treated with rDNase (Macherey-Nagel, Düren, Germany) to digest any contaminating DNA and subsequently purified with the NucleoSpin RNA Clean-up XS kit (Macherey-Nagel)
Label
biotin
Label protocol
Biotinylated cRNA was prepared using the Affymetrix 3’ IVT Express Kit (Affymetrix, Santa Clara, CA, USA) according to the manufacturer’s specifications with an input of 100 ng of total RNA
Hybridization protocol
Per sample, 7.5 µg cRNA of the obtained biotinylated cRNA samples was fragmented and hybridized in a final concentration of 0.0375 µg/µl on the Affymetrix [HT HG U133 + PM96] (Affymetrix, Santa Clara, CA, U.S.A.). After an automated process of washing and staining by the GeneTitan machine (Affymetrix, Santa Clara, CA, U.S.A) using the Affymetrix HWS Kit for GeneTitan (part nr. 901530), absolute values of expression were calculated from the scanned array using the Affymetrix Command Console v3.2 software.
Scan protocol
GeneChips were scanned using the Affymetrix GeneTitan system
Description
fresh frozen Rb material from a randomly selected cohort
Data processing
Data were background adjusted, normalized and summarized by the RMA method. The affy package from Bioconductor was used for this analysis.
