Dept of Clinical Genetics, Lund University Hospital, Sweden
Extracted molecule
total RNA
Extraction protocol
Total RNA was extracted from frozen tumor biopsies using Trizol reagent (Invitrogen, Carlsbad, CA, USA) and RNeasy (Qiagen, Valencia, CA, USA) according to the manufacturers' instructions. RNA quality and concentration were measured using an Agilent 2100 bioanalyzer and Nanodrop ND-1000, respectively. cDNA was generated with the GeneChip® Whole Transcript (WT) cDNA Synthesis and Amplification Kit (Affymetrix) using 300 ng total RNA. Amplified cDNA was fragmented and end labeled using the GeneChip® WT Terminal Labelling Kit (Affymetrix). Subsequently, the fragmented and biotinylated cDNA was hybridized to the GeneChip® Human Gene 1.0 ST Arrays (Affymetrix). The arrays were washed and stained on a GeneChip® Fluidics Station 450 (Affymetrix) according to the manufacturer’s recommendations.
Label
biotin
Label protocol
GeneChip® WT Terminal Labelling Kit (Affymetrix Inc, Santa Clara, CA, USA)
Hybridization protocol
Arrays were hybridized and washed according to the manufacturer's specifications (Affymetrix Inc., Santa Clara, CA, USA)
Scan protocol
Arrays were scanned in a GeneChip® Scanner 3000 (Affymetrix Inc., Santa Clara, CA, USA)
Description
Genome-wide gene expression profiling of MIFS using the Affymetrix GeneChip® Human Gene 1.0 ST Arrays (Affymetrix Inc., Santa Clara, CA, USA)
Data processing
Image analysis was performed using GeneChip® Operating Software (GCOS, Affymetrix). Expression data was normalized, background corrected and summarized using the RMA algorithm implemented in the Affymetrix Expression Console™ version 1.0 software.
