Figure 3

Detection of positive and negative strand HCV RNA in the transfected Huh 7.5 cells by RPA. Huh-7.5 cells were transfected with 10 μg of in vitro transcribed full-length JFH1-GFP and JFH1-GND-GFP mutant HCV RNA by electroporation. Total RNA was isolated from the RNA transfected cell culture at 0, 24, 48, 72 and 96 hours post-transfection. For the detection of positive strand HCV RNA, total cellular RNA was hybridized with a negative strand RNA probe targeted to the highly conserved 5'UTR region and then RPA experiment was performed. For the detection of negative strand RNA, total cellular RNA was hybridized with a positive sense RNA probe targeted to the 5'UTR region and then RPA was performed. (A) Intracellular HCV positive strand RNA in the Huh-7.5 cells transfected with full-length and mutant JFH1-GFP RNA at 0, 24, 48, 72 and 96 hours post-transfection. GAPDH mRNA levels was used as a loading control. (B) Replicative negative strand HCV-RNA in Huh-7.5 cells transfected with JFH1-GFP and JFH1-GND-GFP mutant RNA. The bottom panel shows the intracellular GAPDH mRNA level indicating that equal amounts of RNA were loaded in each well in the RPA assay.