2.3. Detection of BRAF Mutation

Detection of BRAF V600E mutation by ddPCR was performed using 11 μL ddPCR Supermix for Probes (no dUTP, Bio-Rad, Hercules, CA, USA), 1.1 μL ddPCR Mutant Assay BRAF V600E (Bio-Rad) and 9.9 μL (maximum volume) of DNA. Water was used as a ‘no template control’ (NTC) and DNA from a PTC as a positive control.

Droplet digital PCR assays were carried out in a final volume of 20 μl reaction mixture. Droplets were created using the QX200 Droplet Generator (Bio-Rad) and DG8 disposable cartridges. Then, 70 μL of droplet generation oil was placed into the oil well for each sample. The amplification was performed using the GeneAmp PCR System 7900 (Applied Biosystems, Waltham, MA, USA) at the following conditions: 1 cycle of 95 °C for 10 min, 40 cycles of 94 °C for 30 s and 55 °C for 1 min (ramp rate 2.5 °C/s), 1 cycle of 98 °C for 10 min and a 4 °C hold. Analysis of the ddPCR data was performed with the QuantaSoft analysis software, version 1.7.4.0917. Only results with a number of droplets > 10,000 were included in the analysis.