Ddpcr supermix for probes kit

Next Generation Sequencing results were validated using droplet digital polymerase chain reaction (ddPCR) on project RNA samples. First, 100 or 1000 ng samples of RNA were converted to cDNA with the iScript Reverse Transcription kit (BioRad Laboratories, Hercules, CA, USA). Samples were then placed in a thermocycler (BioRad) and run according to kit instructions. Then, the ddPCR Supermix for Probes kit (BioRad) was used to attach probes to cDNA from 5 or 50 ng of total RNA. The reaction mix was then separated into nanodroplets using the Automated Droplet Generator (BioRad). Next, PCR was carried out for 40 cycles per manufacturer’s instructions. Droplets were then analyzed using the QX200 Droplet Reader (BioRad) and data was quantified and copy count calculated using QuantaSoft (BioRad) software.

DNA was isolated from tissues using an AllPrep DNA/RNA kit (Qiagen) according to the manufacturer’s recommendations. Viral DNA was measured in tissues by multiplex qPCR with a multiplex PCR kit (Qiagen) or ddPCR with a ddPCR Supermix for Probes kit (Bio-Rad) using primers in the SIV gag region and the macaque IFN-β gene for sample normalization and cellular quantitation. A Rotor-Gene Q thermocycler (Qiagen) was used for RT-qPCRs, and a QX-100 droplet digital PCR system (Bio-Rad) was used for ddPCRs, as previously described (53 (link), 88 (link)). Samples were assessed by ddPCR when there were less than 10 copies detected by qPCR.

The ddPCR reaction was performed using the QX200 ddPCR system of the Bio-Rad Company, program was 94°C for 10 min, 94°C for 30 s, 58°C for 30 s, 72°C for 30 s, 40 cycles, 98°C for 10 min, and 4°C for storage. The reaction system was configured according to the ddPCR SuperMix for Probes kit (Bio-Rad) to ensure complete mixing and no air bubbles. The droplets were placed into the reader for low signal readings. Sensitivity, specificity, and repeatability experiments were carried out.