Digital droplet PCR

Digital droplet PCR (ddPCR) was performed as secondary platform to quantify integrated vector copy number (VCN) using the same set of RRE/RPL32 primers and probes as for qPCR duplex assay and Bio-Rad ddPCR Supermix for probes (Bio-Rad, Hercules, CA).VCN was determined with the Bio-Rad QX200 droplet digital PCR system as per the manufacturer’s instructions. All the assays worked well according to the minimal information for publication of quantitative digital PCR experiments guidelines²⁰. Two different inputs of genomic DNA (10 and 20 ng per reaction) were used to generate droplets with QX200 droplet generator. Using an Applied Biosystems Veriti 96-well thermal cycler (Life Technologies, Carlsbad, CA), droplets were amplified to end point by heating to 95 °C for 10 min followed by 40 cycles of 95 °C for 30 s and 62.2 °C for 60 s, with a final heating step of 98 °C for 10 min. The plate was then placed into the QX200 droplet reader and data were collected and analyzed using the manufacturer’s QuantaSoft software.