Quantifying HIV-1 Reservoir by RT-qPCR

CD4⁺ T cells (1×10⁶) from ART or ART+GYY4137 treated groups were harvested to isolate total RNA using Qiagen RNAeasy isolation kit and 200 ng of total RNA was reverse transcribed (iScript cDNA synthesis kit, Bio-Rad). Reverse transcribed cDNA was diluted tenfold and amplified using primers against HIV-1 LTRs and seminested—PCR was performed using primers and probe listed in Supplementary file 1f. Serially diluted pNL4.3 plasmid was used to obtain the standard curve. Isolation and RT-qPCR of total HIV-1 DNA were performed as described earlier (Kessing et al., 2017 (link)). Briefly, 1×10⁶ cells were lysed (10 mM Tris-HCl, 50 nM KCl, 400 mg/ml proteinase K) at 55°C for 16 hr followed by inactivation at 95°C for 5 min. Digested product was used as a template to set up the first PCR with Taq polymerase (NEB), 1× Taq buffer, dNTPs, HIV-1 and CD3 primers for 12 cycles. The second-round amplification was done using seminested PCR strategy wherein tenfold dilution of first round PCR product was used as a template, HIV-1, and CD3 primers/probes, Taqman Fast Advance master mix (Applied Biosystems) using SetupOnePlus Real-time PCR system (Applied Biosystems). DNA isolated from ACH2 cells that contain a single copy of HIV-1 per cell was used to obtain standard curve.