Droplet digital PCR

Mutant allele frequency was assessed using the QX200 Droplet Digital PCR (ddPCR) System (BioRad, Milan, Italy) in accordance with the manufacturer’s instructions. The PrimePCR™ ddPCR™ Mutation Assay for humans was used. This kit evaluates EGFR p.E746_A750del and EGFR WT for p.E746_A750del, EGFR p.T790M and EGFR WT for p.T790M, EGFR p.L858R and EGFR WT for p.L858R, KRAS G12X and KRAS WT for G12X, KRAS G13X and KRAS WT for G13X, and KRAS Q61X and KRAS WT for Q61X. ddPCR reaction mixtures contained a final concentration of 250 nM of each of the probes, 450 nM of forward and reverse primers, 1x ddPCR Supermix for Probes (Bio-Rad), and 5∼50 ng DNA in a final volume of 20 µl. Each reaction included a blank sample corresponding to H₂O, another corresponding to wild-type DNA, and a positive control. Fluorescence signals of blank and negative control samples were considered background and used to set up the cut-off. Entire ddPCR reaction volumes were loaded in the appropriate wells of a DG8 cartridge (Bio-Rad) with 70 µl of generator oil (Bio-Rad). Samples were then partitioned into approximately 20,000 water-oil emulsion droplets using the QX200 Droplet generator (Bio-Rad). Forty microliters of the water-oil emulsion were used for the ddPCR reaction that was performed with a C1000 Thermal cycler (Bio-Rad) under 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, and 1 cycle of 98°C for 10 min. After thermal cycling, the plates were transferred to a QX200 Droplet reader. Digital PCR data were analyzed using QuantaSoft analytical software v1.7.4 (Bio-Rad).