In vitro transcription

HCV full-length genome or sub genome containing plasmid DNA (10 μg) was linearized according to the restriction enzyme recognition sequence located at the 3’ end of the viral genome. Linearized plasmid was purified using Qiaprep Spin Miniprep Kit (Qiagen). For T7-based in vitro transcription, 100 pl containing 2 μg plasmid DNA in 60 μl H₂O, 20 μl 5x RRL buffer (1 M HEPES, pH 7.5, 1 M MgCl₂, 1 M Spermidine, 1 M DTT in H₂O), 12.5 μl rNTP solution (25 mM each rNTP), 100 U RNasin ribonuclease inhibitor (Promega) and 60 U T7 polymerase (Promega) were incubated at 37 °C for 2 h. In vitro transcription was boosted by freshly adding 30 U T7 polymerase for further 2 h of incubation time. In vitro transcription reaction was terminated by addition of 7.5 U RQ1 Dnase (Promega) to the reaction mix and incubation for 30 min at 37 °C. In vitro transcribed RNA was purified using the NucleoSpin RNA Clean-up kit (Macherey-Nagel). RNA concentration and purity was evaluated using the NanoDrop One (Thermo Scientific). Quality of the in vitro transcripts was checked by agarose gel electrophoresis.