To prepare cDNA for all assays except the TAR assay [which requires previous polyadenylation for efficient RT of short transcripts (44 (link))], an aliquot of RNA was used in a common RT reaction. A combination of random hexamers and poly-dT was used to avoid bias toward RT of the 3′ end (as can be seen with poly-dT), the 5′ end (as can be seen with random hexamers), or any one gene (as occurs with gene-specific primers); the use of a shorter poly-dT (dT15) with lower annealing temperature helped prevent inhibition of the subsequent ddPCR (44 (link)). This common RT was performed in 50 μl containing 5 μl of 10× SuperScript III buffer (Invitrogen), 5 μl of 50 mM MgCl2, 2.5 μl of random hexamers (50 ng/μl; Invitrogen), 2.5 μl of 50 μM dT15, 2.5 μl of 10 mM deoxynucleoside triphosphates (dNTPs), 1.25 μl of RNAseOUT (40 U/μl; Invitrogen), and 2.5 μl of SuperScript III RT (200 U/μl; Invitrogen). For additional measurement of Pol and Nef transcripts, all reagents were increased proportionally for a final RT volume of 70 μl. Control RT reactions were established using RNA from HIV− donor cells (negative control), “no RT” controls containing patient RNA but no superscript (when RNA yields were sufficiently high), and HIV RNA standards (positive controls; see below). RT reactions were performed in a conventional thermocycler at 25.0°C for 10 min, 50.0°C for 50 min, followed by an inactivation step at 85.0°C for 5 min. For best comparison of different transcripts, aliquots from the same common RT reaction were used in subsequent ddPCR assays for read-through, long LTR, polyA, Tat-Rev, and (in some cases) Pol and Nef regions.