Quantifying HIV DNA in CD4+ T Cells

Digital droplet PCR was performed as previously described (Henrich et al., 2012 (link)). Genomic DNA was extracted from primary non-activated CD4+ T cells using the Gentra Puregene kit (Gentra) following the manufacturer’s instructions. For each PCR reaction, 5 units of restriction enzyme BsaJI (NEB) was directly mixed with 300ng of DNA, ddPCR Supermix for Probes (Bio-Rad), and final concentrations of 900nM primers and 250nM probe. Primers/Probes were: RPP30 – forward primer GATTTGGACCTGCGAGCG, reverse primer GCGGCTGTCTCCACAAGT, probe VIC-CTGAACTGAAGGCTCT-MGBNFQ; HIV-gag – forward primer TACTGACGCTCTCGCACC, reverse primer TCTCGACGCAGGACTCG, probe FAM-CTCTCTCCTTCTAGCCTC-MGBNFQ. Droplets were prepared using the QX100 Droplet Generator (Bio-Rad) following the manufacturer’s instructions. Sealed plates were cycled using the following program: 95°C for 10 min; 40 cycles of 94°C for 30 s, 60°C for 1 min; and 98°C for 10 min, with 2°C/sec ramping speed to ensure even droplet heating. Reactions were analyzed using the QX100 Droplet Reader and number of template molecule per μl of starting material was estimated using the Quantalife ddPCR software. We aimed to run 8 replicates of ddPCR per sample – depending on the amount of DNA availability. We consistently applied a pre-determined exclusion criterion to outliers that deviated from mean values by > 2x the standard deviation.