Array-CGH Analysis of CRC Samples

DNA isolated from each of the CRC cultures and their corresponding PBTs were simultaneously profiled for copy number using an oligonucleotide array-CGH platform (SurePrint G3 Human CGH Microarray 8x60K (Agilent Technologies, CA). DNA isolated from peripheral blood from multiple normal individuals was used as reference. Digestion, labeling and hybridization were performed according to our previous protocols [16 (link), 17 (link)]. Briefly, equal amounts of CRCs (and PBTs) and reference DNA, were enzymatically digested and directly labeled with SureTag Labeling Kit (Agilent Technologies, CA). The labeled DNA was hybridized with human Cot1-DNA (Life Technologies, CA) to the arrays, at 65°C for 40 hours. The scanned data was analyzed using the Feature Extraction (FE) software v.10.10 following importing into Agilent Cytogenomics v.2.9.2.4 software (Agilent Technologies, CA). The algorithm ADM-2 and a threshold value of 6.0 were applied with the appropriated filters to analyze the data. Gene amplifications and deletions were considered when the corresponding plotted oligo-probes presented values of log2 ≥7/6 and log2≤5/6, respectively. Duplicate experiments were performed independently for both the CRCs and corresponding PBT to assess data reproducibility.