Total RNA was extracted from frozen tumor biopsies using Trizol reagent (Invitrogen, Carlsbad, CA, USA) and Rneasy (Qiagen, Valencia, CA, USA) according to the manufacturer's recommendations. RNA quality and concentration were measured using an Agilent 2100 bioanalyzer and Nanodrop ND-1000, respectively. cDNA was generated with the GeneChipR Whole Transcript (WT) cDNA synthesis and Amplification Kit (Affymetrix) using 300 ng total RNA. Amplified cDNA was fragmented and end-labelled using the GeneChipR WT terminal labelling Kit (Affymetrix). Subsequently, the fragmented and biotinylated cDNA was hybridized to the GeneChipR Human Gene 1.0 ST Arrays (Affymetrix). The arrays were washed and stained on a GeneChipR Fluidics Station 450 (Affymetrix) according to the manufacturer's recommendations.
Label
Biotin
Label protocol
GeneChipR WT Terminal Labelling Kit (Affymetrix Inc, Santa Clara, CA, USA)
Hybridization protocol
Arrays were hybridized and washed according to the manufacturer's instructions (Affymetrix Inc, Santa Clara, CA, USA).
Scan protocol
Arrays were scanned in a GeneChipR scanner 3000 (Affymetrix Inc, Santa Clara, CA, USA).
Description
Gene expression analysis for breakpoint mapping.
Data processing
Image analysis was performed using GeneChipR Operating Software (GCOS, Affymetrix). Expression data were normalized, background-corrected and summarized using the RMA algorithm implemented in the Affymetrix Expression Console version 1.0 software.
