Polyadenylation for Efficient HIV Transcript Detection

Reverse transcription from a linker molecule (which we achieve using polyA polymerase to attach a polyA tail) is necessary for efficient reverse transcription of short, prematurely-terminated HIV transcripts limited to the TAR loop [31 (link)]. Therefore, a polyadenylation step was employed prior to reverse transcription and ddPCR for the TAR region [27 (link), 31 (link)]. Each polyadenylation reaction comprised cellular RNA with 3μL of 10x Superscript III buffer (Invitrogen), 3μL of 50mM MgCl₂, 1μL of 10mM ATP (Epicentre), 2μL of 4U/μL poly-A polymerase (Epicentre), and 1μL of 40U/μL RNaseOUT (Invitrogen) in a 20μL reaction. The reaction was incubated at 37μC for 45min prior to addition of RT reaction components, including 1.5μL of 10mM dNTPs (Invitrogen), 1.5μL of 50ng/μL random hexamers (Invitrogen), 1.5μL of 50μM oligo dT15, and 1μL of 200U/μL Superscript III reverse transcriptase (Invitrogen). Reverse transcription was performed on the final 30μL reaction 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.