Genomic DNA was isolated from 10 10 m-thick paraffin-embedded tissue sections. Sections were deparaffinated twice for 5 min in xylene, rehydrated in 100, 96 and 70% ethanol for 30 s each, stained with haematoxylin for 30 s, rinsed with water and incubated overnight in 1 m NaSCN at 37°C to remove crosslinks. Slides were rinsed twice 10 min in 1 PBS at room temperature, and completely air-dried. Tumour tissue was scraped from the glass with a scalpel to obtain at least 70% tumour cells in 200 l Qiagen ATL buffer (QIAamp® DNA extraction kit cat. 51306), transferred to eppendorf tubes and incubated with 27 l proteinase-K (20 mg/ml stock) at 450 rpm (Eppendorf® Thermomixer R) at 55°C. Three more aliquots of 27 l proteinase-K were added at 4, 20 and 28 h. After a total protK incubation of 44 h, DNA isolation proceeded as in the manufacturer's protocol (Qiagen, Cat. 51306). Samples of isolated genomic DNA were analysed by 0.8% agarose gel electrophoresis to visualise DNA concentration and size distribution. In case of tumour tissue, we scraped regions containing at least 70% tumour as indicated by an experienced breast cancer pathologist.
Label
Cy3
Label protocol
Labeling was performed using the Enzo Genomic DNA Labeling kit according to the manufacturer's instructions (Enzo Life Sciences). Briefly, 500 ng genomic DNA was combined with a mixture of primers and reaction buffer to a final volume of 39 μl. The DNA was denatured at 99°C for 10 min, and placed on ice. While on ice, 10 μl cyanine 3-dUTP and cyanine 5-dUTP nucleotide mixture was added to the test and reference DNA, respectively, and finally 1 μl Klenow DNA polymerase was added to each sample to extend the primers. After 4 hr incubation at 37°C, 5 μl stop buffer was added to stop the reaction. Label was purified using the QIAquick PCR Purification Kit (Qiagen, Westburg, Leusden, NL).
aCGH reference DNA was isolated from peripheral blood lymphocytes from six apparently healthy female individuals. It was pooled and sonicated until its median fragment length was similar to that of the test samples.
Label
Cy5
Label protocol
Labeling was performed using the Enzo Genomic DNA Labeling kit according to the manufacturer's instructions (Enzo Life Sciences). Briefly, 500 ng genomic DNA was combined with a mixture of primers and reaction buffer to a final volume of 39 μl. The DNA was denatured at 99°C for 10 min, and placed on ice. While on ice, 10 μl cyanine 3-dUTP and cyanine 5-dUTP nucleotide mixture was added to the test and reference DNA, respectively, and finally 1 μl Klenow DNA polymerase was added to each sample to extend the primers. After 4 hr incubation at 37°C, 5 μl stop buffer was added to stop the reaction. Label was purified using the QIAquick PCR Purification Kit (Qiagen, Westburg, Leusden, NL).
Hybridization protocol
Labelled DNA was hybridized on a 720K Nimblegen CGH array (human CGH 3x720k whole-genome tiling v3.1 arrays: GPL14965 and GPL15436), containing 719690 unique in situ synthesized 60-mer oligonucleotides, according to manufacturer’s protocol (Roche Nimblegen, Madison, USA).
Scan protocol
Slides of the arrays were scanned using a G2505C microarray scanner (Agilent Technologies).
Description
444777A02
Data processing
Data was processed using the NimbleScan sorftware provided by the vendor. The RATIO_CORRECTED value, a background corrected log2 ratio of the two color channels, was used in all subsequent analyses.
