Genomic DNA was purified from frozen samples following the DNeasy Blood and Tissue Kit (Qiagen, Valencia, CA). Briefly, 0.25 mg of frozen tissue was placed in Qiagen buffer and Proteinase K (>600 mAU/ml) and incubated for 3 hours at 150 rpm. Following tissue lysis and protein removal, genomic DNA was eluted in 100µl of Buffer AE (Qiagen). Only samples with a spectrophotometric absorption ratio of 260/280 > 1.8 (Nanodrop ND-1000 spectrophotometer, NanoDrop , Wilmington, DE) were included in subsequent assays. Purified genomic DNA was evaluated using agarose gels (2%) or microfluidic chips (12000 DNA Chip, Bioanalyzer 2100, Agilent Technologies, Santa Clara, CA) to confirm that the DNA from each specimen was of high molecular weight (> 10Kb) with minimal detectable degradation.
Label
biotin
Label protocol
Sty I and Nsp I restriction enzymes (New England Biolabs, Ipswich, MA) were used to separately digest 250ng of DNA resulting in a total digest of 500 ng of whole genomic DNA per sample. Each sample was ligated with the appropriate adaptor (Sty or Nsp) provided in the Affymetrix 6.0 protocol and a total of seven PCR reactions were performed per sample (three Sty I restriction digest/ligation DNA reactions and four Nsp I restriction digest/ligation DNA reactions) and pooled into one tube with a total volume of 700µl. Purification of resulting PCR products was performed using the protocol associated with the Affymetrix Cytogenetics Copy Number Assay which required the use of Agencourt AMPure beads (Beckman Coulter, Danvers, MA). Each pooled sample was fragmented with DNAse1, biotinylated and end-labeled.
Hybridization protocol
Samples were hybridized on Genome-Wide SNP 6.0 arrays for 18 hours at 500C with rotation (60 rpm) in an Affymetrix Gene Chip Hybridization Oven (Model 640).
Scan protocol
Arrays were washed, stained and scanned according to the Affymetrix Genome-Wide SNP 6.0 protocol using the Affymetrix GeneChip Fluidics Station (Model 450), GeneChip Scanner 3000 7G, and GeneChip Command Console (v3.0.2) software.
Data processing
The Affymetrix Genotyping Console (v3.0.2) was used to generate a SNP call for each probe set on each array. SNP calls were determined for an average of 96.78% (S.D.= 4.27%) of the array probe sets per specimen throughout the study. Affymetrix CEL and CHP files were transferred to Partek Genomics Suite v6.5 (Partek Inc, St Louis, MI) for copy number and loss of heterozygosity (LOH) analysis respectively. CEL files were imported using the default setting, which adjusts hybridized intensities based on fragment length and probe sequence. Copy number was generated by comparing the hybridized intensities of each array to the laboratory normal tissue reference set (n=31) eliminating inter-laboratory variability introduced by comparison to other databases such as the HapMap. Copy number measurements were smoothed based on local GC content using a 1 Mb window. Partek segmentation algorithm was used to identify regions of significant copy number variation (CNV) from normal consisting of a minimum of 30 genomic markers and p-value of less than .001, with signal to noise and expected range set to 0.3. The median length of inter-marker spacing on the SNP 6.0 array is 680 bp with a median length of a 30 marker region approximately 20Kb. Regions of LOH were identified for tumors with a matched normal pair (2 chromophobe, 1 clear cell, 1 papillary type 1, and 1 papillary type 2 sample) using the Hidden Markov Model in Partek Genomics Suite. There is copy number data for each tumor sample supplied in the matrix. This copy number data was created using the laboratory unpaired normal reference set (available in CEL file format) and has been GC corrected.
