Reverse Transcription. Total RNA (500 ng) was reverse-transcribed in a volume of 40 µl (Table 2.3). RNA extracts were first incubated with 0.2 µg Random Hexamer (Bioline) at 70 °C for 5 minutes and allowed to cool down to room temperature. A master mix containing 5X Reaction Buffer (MBI Fermentas), dNTP mix (10 mM; MBI Fermentas), and Ribolock Inhibitor (40 u/µl; Fermentas) was then added and the mixture incubated at room temperature for 5 minutes. Revert-Aid™ Reverse Transcriptase (200 u/reaction; Fermentas Life Sciences) was then added and the reaction mix subsequently incubated using a PTC-200 Thermal Cycler (MJ Research) at room temperature for 10 minutes; 42 °C for 1 hour; and then at 70 °C for 10 minutes to stop the reaction. The reverse transcription reaction products were 10-fold diluted to 400 µl to be within the linear range of PCR amplification. Reagent Volume RNA 500ng Random Hexamer 8 µl Deionised water Upto 25 µl 5X Reaction Buffer 8 µl dNTP mix 4 µl Ribolock Inhibitor 0.5 µl Deionised water 1.5 µl Revert-Aid™ Reverse Transcriptase 1 µl Total 40 µl Table 2.3 Volumes of each reagent added to reaction.
Reverse Transcription. After entering a host cell, the protein core disintegrates releasing viral proteins and two RNA copies of the viral genome. The HIV RT binds to the viral RNA and converts the single-stranded RNA into double-stranded DNA.15 The single-stranded viral RNA is used as a template by the RT to synthesize single-stranded DNA.15 As this polymerization takes place, the RNA template is degraded by HIV RT’s second enzymatic active site, the ribonuclease H (RNase H) active site.16 The newly synthesized single-stranded viral DNA is then used as a template by HIV RT to produce double- stranded viral DNA.15 The double-stranded DNA is now a substrate for the viral integrase and can now be integrated into the host cell’s DNA.15
