Ribonuclease protection assay and Real-time RT-PCR of sub-genomic HCV 1b RNA. (A) Intracellular expression of IRF9-S1C and IRF9-S2C or both in R15-3 cells inhibits HCV negative strand RNA detection by RPA. R15-3 cells were transfected treated (-) and (+) IFN-α (1,000 IU/ml), and total RNA was isolated at 72 hours post-transfection. The upper panel shows the RPA results of HCV negative strand detection. The bottom panel shows GAPDH mRNA levels by RPA. (B) Real-time RT-PCR was performed to quantify HCV RNA in R15-3 transfected cells. Cellular RNA was isolated at 72 hours, retrotranscribed, and assayed by real time PCR. S9-13 and R15-3 cells (+) and (-) IFN-α were used as (+) and (-) controls, respectively. Error bars represent SD. Asterisk (*) indicates transfected R15-3 cells with significantly lower HCV RNA relative to untransfected R15-3 cells treated with IFN. Significance was considered at p-values < 0.05.