All samples were normalised using a global median normalization to obtain log2-ratios as described in Buffart et al. Genes Chromosomes & Cancer: 47(11);994-1004 (2008).
A wave smoothing correction was performed on the normalised data as described in: 'Smoothing waves in array CGH tumor profiles'; Van de Wiel et al. Bioinformatics:25(9);1099-1104 (2009).
Following hybridization, the arrays were scanned using the MS 200 Microarray Scanner (Roche NimbleGen), according to the manufacturer’s instructions (NimbleGen Arrays User’s Guide – CGH and CNV Arrays, version 8.1).. (Parameters: Scanning hardware = OTHER: MS200 Microarray Scanner (Roche), Scanning software = Scanning software)
The samples were purified using the MinElute PCR Purification Kit (Qiagen). Hybridization was performed using the manufacturer’s instructions (NimbleGen Arrays User’s Guide – CGH and CNV Arrays, version 8.1). (Parameters: Chamber type = OTHER: NimbleGen Hybridization System, Quantity of label target used = 1, Mass unit = Micro gram, Tiny time unit = seconds, Volume unit = Nano litre, temperature = 42)
Labeling was performed using the CGH Labeling Kit for Oligo Arrays (Enzo Life Sciences, Farmingdale, NY, USA) as follows: 1 ug DNA from the tumor and a human male or female reference (Kreatach Diagnostics, Amsterdam, The Netherlands) was denatured together with 20 ul random primers in reaction buffer for 10 min at 98 degrees C and placed on ice (4 degrees C) for 5 min. Next, primer extension was performed using 10 ul nucleotide mixes containing either Cyanine 3-dUTP or Cyanine 5-dUTP nucleotide mixes and 1 ul Klenow Exo-DNA polymerase were added while the samples were still at 4 degrees C. The tumor samples were labeled with Cyanine 3-dUTP and the human reference samples with Cyanine 5-dUTP. After mixing, the samples were incubated for 4 hours at 37 degrees C. The labeling reaction was terminated by adding the Stop Buffer to the mixture and cooling down to 4 degrees C. See also Buffart et al. Genes Chromosomes & Cancer: 47(11);994-1004 (2008). (Parameters: Amount of nucleic acid labeled = 1, Amplification = none, Mass unit = Micro gram)
DNA was isolated using the QIAamp DNA MiniKit (Qiagen, Hilden, Germany), according to the manufacturer's instructions. (Parameters: Extracted product = genomic_DNA, Amplification = none) Male and female reference genomic DNA samples were also prepared according to manufacturer's instructions.
All samples were from resected tumors that were fresh frozen. From these frozen tumors, sections were cut. All sections contained >70% tumor cells, as verified by a pathologist. Genomic DNA was later isolated using thse sections.