Quantasoftware

Genomic DNA extraction and isolation from EDTA blood were performed with the MagNA Pure 96 system using DNA Tissue Lysis Buffer and viral NA Small RNA kit (Roche, Basel, Switzerland) according to manufacturers´ manual. Similar to that described by Bannasch et al. [15 (link)], the copy number quantification of the KITLG CNV was performed with digital droplet PCR (ddPCR) using TaqMan^® assays specific for the KITLG CNV sequence and proto-Oncogene 1 (ETS1) as reference gene. Measurement was carried out in duplicate, and the mean value was used for further analyses. Intra-assay correlation was 0.85. Copy number was determined using DropletReader (Bio-Rad, Feldkirchen, Germany) and QuantaSoftware (Bio-Rad, Feldkirchen, Germany).

Extracted genomic DNA was restriction digested with the EcoRI enzyme (New England Biolabs) for 1 h at 37 °C. A 20-µL PCR mixture solution containing 1× ddPCR SuperMix (Bio-Rad), 1× probe and primer premix for the target gene and internal control gene, RNase P (final concentration of 250 nM for the probe and 900 nM for each primer; Applied Biosystems), and 10 ng of the restriction digested DNA was prepared. The reaction mixture and droplet generation oil (Bio-Rad) were loaded into a droplet generator (QX-200; Bio-Rad). The droplets were transferred to a 96-well PCR plate, and PCR was performed as follows: enzyme activation for 10 min at 95 °C; 40 cycles of 94 °C for 30 s, 60 °C for 1 min, and 98 °C for 10 min; enzyme deactivation for 10 min at 98 °C; and a hold at 4 °C (performed with a ramp rate of 2 °C/s in all steps). The PCR plate was placed in a droplet reader (Bio-Rad). After the reading, the copy number variation of each target gene was analyzed with Quanta software (Bio-Rad) on the droplet reader. The amplification threshold value was set at 2.5 for patient tissue specimens and 3.0 for cell lines.