Array-CGH Analysis of DNA Copy Number Alterations

DNA copy number alterations (CNAs) were evaluated by array-CGH analysis using the Agilent platform (Agilent Tech., Santa Clara, CA, USA) as previously described [93 (link)]. Briefly, equal amounts of the isolated tumor and reference genomic DNA (a pool obtained from multiple female individuals with no cancer) (100–300 ng) were digested and labeled using a SureTag Complete DNA Labeling Kit (Agilent Tech.) and hybridized in the arrays for 40 h. Only cases that showed satisfactory incorporation of more than 1.00 pico/mol labeling were selected for hybridization. The array data were extracted using Feature Extraction (FE) software v10.10, and the Agilent Cytogenomic v. 7.0 software (Agilent Tech.) was used to analyze the data using the aberration detection method-ADM2, a threshold of 6.0, and defined aberration filters. Copy number gains and losses were considered when present in at least 3 consecutive probes with values of mean absolute log2 ratio (intensity of the Cy5 dye (reference DNA)/intensity of the Cy3 dye (test DNA) value of ≥0.25 and ≤−0.25, respectively) as per our previous analysis [20 (link)]. UCSC Genome Browser (GRCh37/hg19) and miRbase 22.1 databases were used to determine the genes and miRNAs present in each selected cytoband affected by CNA, respectively.