A small piece of frozen tissue was removed from a sample of frozen tissue and incubated in a proteinase K digestion solution followed by cleanup using the Qiagen M48 robotics protocol.
Label
biotin
Label protocol
Following restriction enzyme digestion, biotin labeled DNA was prepared according to the Affymetrix protocol from 500ng of starting DNA (GeneChip Mapping 500K Assay Manual, Affymetrix, 2005). The fluidics station model FS450 was used.
Hybridization protocol
Mapping 500K arrays were hybridized according to the Affymetrix protocol (GeneChip Mapping 500K Assay Manual, Affymetrix, 2005) using an Affymetrix model 640 hybridization oven.
Scan protocol
Mapping 500K arrays were scanned according to the Affymetrix protocol (GeneChip Mapping 500K Assay Manual, Affymetrix, 2005) using a GS3000 scanner.
Description
NA
Data processing
Affymetrix 500K Mapping Array intensity signal CEL files were processed by dChip 2005 (Build date Nov 30, 2005) using the PM/MM difference model and invariant set normalization. Forty-eight normal samples were downloaded from the Affymetrix website (http://www.affymetrix.com/support/technical/byproduct.affx?product=500k) and analyzed at the same time. One CEL file for each set (Sty and Nsp) with the median signal intensity across the set was selected as the reference array. The dChip-normalized signal intensities were converted to log2 ratios and segmented as follows. For each autosomal probe set, the log2 tumor/normal ratio of each tumor sample was calculated using the average intensity for each probe set in the normal set. For Chromosome X, the average of the 20 normal female samples was used. Each probe set was mapped to the genome, NCBI assembly version 35, using annotation provided by the Affymetrix web site. The log2 ratios were centered to a median of zero and segmented using the GLAD package for the R statistical environment.
