Digital PCR (dPCR)

dPCR was carried out using Qx200 ddPCR EvaGreen Supermix (Bio-Rad) according to the manufacturer’s instructions. Specifically, a 20-µL mixture containing AP3B1 primers or MYU primers (Table 1), the cDNA sample, premix (2X), and H₂O was prepared for each reaction. Triplicate reactions were prepared for each cDNA sample. The 20 µL mixture was loaded into the sample wells of a DG8 Cartridge (Bio-Rad), followed by 70 µL of QX200 Droplet Generation Oil for EvaGreen (Bio-Rad) into the oil wells. Then, the DG8 Cartridge was put into the QX200 Droplet Generator (Bio-Rad) to form droplets which were subsequently transferred into a 96-well PCR plate (Eppendorf). The amplification was performed on the Eppendorf Mastercycler NEXUS (Eppendorf) and the thermal cycling conditions were: 95 °C 5 min, denaturation 95 °C 30 s, annealing/extension 60 °C 1 min, 40 cycles. After the amplification, the plate was put into the QX200 Droplet Reader (Bio-Rad) where the droplets from each well of the plate were read automatically. A five-point standard curve and a negative control were used in all runs.