RT-ddPCR Protocol for HIV-1 mRNA Quantification

For RT-ddPCR, cDNA was synthesized from 500 ng of RNA and a 20 μL reaction mixture with the following components: 4 μL of 5× 1st strand buffer (Invitrogen, Waltham, MA, USA), 2 μL of 0.1 M DTT (Invitrogen, Waltham, MA, USA), 2 μL of random hexamers (50 μg/mL; Promega, Madison, WI, USA), 1 μL of 10 mM deoxynucleoside triphosphates (dNTPs) mix, 0.1 μL of RNaseOut (40 U/μL; Invitrogen), 0.5 μL of SuperScript II RT (200 U/μL; Invitrogen, Waltham, MA, USA) and nuclease-free water to bring the final reaction volume to 20 μL. The reverse transcription reaction was carried out at 42 °C for 50 min followed by at 70 °C for 15 min. Negative controls containing no SuperScript II RT were also included in the reaction [42 (link)]. RT-ddPCR was performed using Bio-Rad QX200 AutoDG Digital Droplet PCR system. Primer sets used for the amplification of HIV-1 mRNA are detailed in Table 4.