Qiagen’s QIAamp DNA Micro kit (Qiagen, Hilden, Germany)
Label
Cy5
Label protocol
Labelling 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).
Qiagen’s QIAamp DNA Micro kit (Qiagen, Hilden, Germany)
Label
Cy3
Label protocol
Labelling 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
Cy3- and Cy5-labeled DNA samples were combined in a total volume of 79 μl and mixed with 6.5 μg Cot-1 DNA (Invitrogen), 25.5 μl of 10× blocking agent (Agilent Technologies), 130 μl of 2× hybridization buffer (Agilent Technologies), and 15.5 μl MilliQ water. The hybridization mixture was heated for 3 min at 95°C and immediately incubated for 30 min at 37°C. After centrifugation for 60 sec at 14,000 rpm, the hybridization mixture was applied to the 2x 105K array slide and placed in an assembly chamber for 24 hr at 65°C and 20 rpm (Agilent Technologies). After hybridization, slides were washed in the following steps; 1 min with wash buffer 1 at room temperature (RT), 5 min with wash buffer 1 at RT with rotation speed (750 rpm), 1 min with wash buffer 2 at 37°C with rotation speed (750 rpm), and finally a 1 min wash step with acetonitrile at RT.
Scan protocol
Images of the arrays were acquired using a microarray scanner G2505B (Agilent technologies) and image analysis was performed using feature extraction software (version 9.1; Agilent Technologies). The Agilent CGH-v4_91 protocol was applied using default settings. Spot intensities were corrected for local background, and only spots with intensities at least twofold above the background signal intensities were included in the analysis.
Data processing
image analysis was performed using feature extraction software (version 9.1; Agilent Technologies). Spot intensities were corrected for local background, and only spots with intensities at least twofold above the background signal intensities were included in the analysis. Two samples were excluded based on their noisy profiles and high Median Absolute Deviation value (>0.5). The data from the remaining 47 samples had their background deducted and were normalized by median centering. Inherent wave bias using the Smoothing Waves algorithm was used to correct the profiles [35]. The data was then segmented using the DNACopy algorithm to find fragments of copy number increase or decrease [36]. Copy number states were deduced by using the CGHCall algorithm [37]. 85% tumor tissue was specified as parameter for the calling and for all other parameters default values was used. The oligonucleotides were mapped according to the human genome build NCBI 36 for the 2 × 105 K array format. Of both Cy3 and Cy5 channels, local background was subtracted from the median intensities. The log2 tumor to normal ratio was calculated for each spot and normalized against the median of the ratios of all autosomes. When two or more neighbouring oligonucleotides showed significant copy number differences from zero, this was considered to be a possible CNA (copy number aberration).
