Sureprint g3 human cgh microarray 8x60k

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.

DNA copy number analysis was performed using an oligonucleotide a-CGH platform (SurePrint G3 Human CGH Microarray 8x60K; Agilent Technologies Inc., Santa Clara, CA, USA), using a previously established protocol for FFPE samples [43 (link), 44 (link)]. DNA was isolated using the standard phenol-chloroform method. Reference DNA was prepared from the peripheral blood of a pool of ten healthy donors [45 (link)]. Equal amounts of tumor and reference genomic DNA (1–2 μg) were digested, enzymatically labeled using the SureTag Complete DNA Labeling Kit (Agilent Technologies, Inc., Santa Clara, CA, USA), and hybridized to the arrays. The array data were analyzed with the Feature Extraction v.10.10 software and Agilent CytoGenomics v.3.0 software (Agilent Technologies Inc., Santa Clara, CA, USA) using the ADM-2 algorithm, threshold 6.0, and an aberration filter with a minimum of >3 probes [45 (link)]. Copy number gains and losses were defined as previously described [44 (link)].

Genomic DNA extraction, sample preparation, slide hybridization and analysis were performed using SurePrint G3 Human CGH Microarray 8x60K (Agilent Technologies, Santa Clara, CA, USA) following the manufacturer’s recommendations. The arrays were scanned at 2-μm resolution and analyzed using Feature Extraction v10.7 and Agilent Genomic Workbench v5.0 software (Agilent Technologies), as previously reported [9 (link)].