Agilent genomic workbench

Microarray data were analyzed using Agilent Genomic Workbench (Agilent Technologies, Santa Clara, CA) and BRB-arraytools (http://linus.nci.nih.gov/BRB-ArrayTools.html). Agilent Genomic Workbench was used to calculate log₂^ratio for every probe and to identify genomic aberrations. Mean log₂^ratio of all probes in a chromosome region between 0.25 and 0.75 was classified as genomic gain, >0.75 as high-level DNA amplification, <−0.25 as hemizygous loss, and <−0.75 as homozygous deletion. In pathway enrichment analysis, p-value is calculated for each pathway based on the null distribution obtained by a 1000-time random sampling method.

Array comparative genomic hybridization was carried out using the SurePrint G3 Human CGH Microarray 8 × 60 K (Design ID 021924, Agilent Technologies). DNA isolation of the cell lines was performed using the QiAmp DNA Mini Kit (Qiagen). Quantity and quality of the DNA were assessed on the NanoDrop platform (Thermo Fisher Scientific, Rockford, USA). DNA hybridization was performed according to the standard procedures after labelling of 500 ng of the sample DNA and the control DNA (Human Reference DNA Female or Human Reference DNA Male, Agilent Technologies). Microarray data was analysed using Agilent Genomic Workbench (Agilent Technologies).

Copy number alterations were analyzed by microallelotyping using polymorphic dinucleotide microsatellite markers D5S642 and D5S2057, located 0.6 Mb centromeric and 1.8 Mb telomeric of ADAMTS19. In some cases where both markers were in homozygosis, we also analyzed D5S2098, located 5 Mb upstream of ADAMTS19. Primer sequences to amplify these markers were obtained from the Ensembl website [61 (link)]. PCR amplification was performed in presence of α-³²P-dCTP and resolved in vertical electrophoresis acrylamide-bisacrylamide gels. After electrophoresis, gels were dried and exposed to X-ray films. Loss of heterozygosity was assessed in heterozygous cases by the relative change in intensity in one of the bands when comparing the normal and tumor sample. aCGH was performed using Agilent 44K arrays, following the manufacturer’s protocol. Copy number alterations were analyzed using Agilent Genomic Workbench, with ADM-2 algorithm, threshold of 6, and Fuzzy Zero correction. Only alterations with a minimum of three consecutive probes were considered valid.