Abi copycaller 2

Copy number variation was evaluated on 20 ng of genomic DNA. Quantitative real-time polymerase chain reaction (PCR) TaqMan Copy Number Assays were performed using three probes targeting different regions of the E2F1 gene (Hs00576444_cn, Hs01758822_cn and Hs00919582_cn)(Applied Biosystems, Foster City, CA, USA). TaqMan CNV reactions were performed in triplicate using the FAM-dye-labeled assay for E2F1 and VIC-dye labeled RNase P assay. Real-time data were collected by the StepOne Plus 2.1 software, and ABI CopyCaller 2.0 software (Thermo Fisher Scientific Inc, Waltham, MA, USA) was used for data analysis. Two independent assays were performed for each sample to confirm results.

Genomic DNA of patients was provided by the above-mentioned biobank. DNA was isolated from peripheral blood leucocytes using QIAamp DNA Blood Mini Kit, according to the manufacturer’s protocol (Qiagen Inc., Hilden, Germany). Copy number variation was evaluated on 20 ng of genomic DNA. Quantitative real-time polymerase chain reaction (PCR) TaqMan Copy Number Assays were performed using three probes targeting different regions of the E2F1 gene (Hs00576444_cn, Hs01758822_cn and Hs00919582_cn)(Applied Biosystems, Foster City, CA, USA). TaqMan CNV reactions were performed in triplicate using the FAM-dye-labeled assay for E2F1 and VIC-dye labeled RNase P assay. RNase P assay was used to normalize the genomic DNA input.
An internal DNA resulted with two copies of E2F1 both by TaqMan Copy Number Assay and array CGH was used as calibrator. Real-time data were collected by the StepOne Plus 2.1 software, and ABI CopyCaller 2.0 software (Thermo Fisher Scientific Inc, Waltham, MA, USA) was used for data analysis. Copy Number ranging from 1.5 to 2.5 were predicted as CNV = 2. Two independent assays were performed for each sample to confirm results.