Dna analytics software

Comparative genomic hybridization array (aCGH) analysis was performed using Agilent Human Genome CGH Microarray 60K kit (Agilent Technologies, Palo Alto, CA, USA), following the manufacturer’s instructions. A sex-matched commercial DNA sample (Male, Promega, Milan, Italy) was used as reference DNA. Hybridization signals were analyzed using Feature Extraction software (v10.7) and DNA Analytics software (v5.0, Agilent Technologies, Palo Alto, CA, USA). Aberration Detection Method 2 (ADM2) algorithm (threshold 5.0) was used to identify DNA copy number aberrations. We applied a filtering option of a minimum of three aberrant consecutive probes (Wu et al., 2007 (link)) and a minimum absolute average log 2 ratio of 0.30. University of California Santa Cruz (UCSC) human genome assembly hg18 was used as a reference and copy number variations (CNVs) were identified with a database integrated into the Agilent Genomic Workbench analytic software. Log two ratios lower than −0.30 were classified as losses, those greater than 0.30 as gains. The analysis revealed CNVs for the one iPSC clone, consisting of amplification of 36 kb and 175 Kb, on chromosomes 2 and 3, respectively, involving genes with no significant associated clinical phenotype. The clone from healthy control did not reveal any CNVs (Table 5).

DNA (1.25 μg) samples from normal ear and mammary carcinoma samples were labeled with cyanine 3- and cyanine 5-dUTP, respectively, and purified using columns (Agilent Technologies, Santa Clara, CA, USA). Labeled DNA was hybridized with microarray probes (Rat CGH, 2×105K; Agilent) at 65°C with rotation at 20 rpm for 40 h, and then washed with Wash Buffers 1 and 2 (Agilent). The microarray resolution was ~14.5 kb (on average) with 97,973 probes, which were annotated in the rn4 version of assembly. Microarrays were scanned using the Agilent G2565BA microarray scanner. Fluorescence intensity values were obtained from scanned images with Agilent Feature Extraction software (ver. 9.5.1, Agilent) and were analyzed using DNA Analytics software (ver. 4.0.81, Agilent). Rat orthologs of human genes relevant to breast cancer [27 (link)] were identified in the rn4 rat genome assembly using the UCSC Genome Browser (https://genome.ucsc.edu/) [28 (link)]. Annotations pertaining to the role of genes in cancer were retrieved from the Oncogene Database (http://ongene.bioinfo-minzhao.org/), Tumor Suppressor Gene Database (https://bioinfo.uth.edu/TSGene/), and COSMIC Database (https://cancer.sanger.ac.uk/census). Microarray data are available at the Gene Expression Omnibus database (https://www.ncbi.nlm.nih.gov/geo/; accession number GSE160514).

For each experiment, purified FFPE DNA (500 ng) and reference DNA (500 ng) (NA10851; Coriell Institute for Medical Research, Camden, NJ, USA) were differentially labeled with cyanine 5 (Cy5) and cyanine 3 (Cy3) fluorescent dyes using Universal Linkage System technology (Agilent Technologies), a non-enzymatic direct labeling method. Unreacted dye was removed using KREApure purification columns (Agilent Technologies). DNA labeling efficiency was assessed by NanoDrop 2000 spectrophotometry (ND-2000; Thermo Fisher Scientific, Waltham, MA, USA). The degree of labeling (DoL, the number of fluorophore molecules per 100 nucleotides) was calculated using post-labeling DNA yield and fluorophore concentration. DoL values ranging from 0.75% to 2.5% and 1.75% to 3.5% were considered optimal for Cy5- and Cy3-labeled DNA, respectively. Cy5- and Cy3-labeled DNA hybridization was performed using dual-color array containing 60-mer oligonucleotide probes (SurePrint G3 Human CGH Microarray 8 × 60 K; Agilent Technologies). Following hybridization, the arrays were scanned using a Dual Laser Microarray Scanner (G2565CA; Agilent Technologies). The images were extracted and analyzed using Feature Extraction software version 10.5.1.1 (Agilent Technologies) and DNA Analytics software version 4.0.73 (Agilent Technologies).