FIG. 4.
(A) Schematic of the RPA. A radiolabeled RNA probe contains 377 nucleotides (nt) complementary to either the positive-strand 5′ UTR or negative-strand 3′ UTR of the JFH1 HCV (genotype 2a) and an additional 40 unpaired nucleotides derived from the vector. After hybridization and digestion with RNase A/T1, the region (377 nucleotides) protected from RNase digestion migrates faster than the incompletely digested RNA probe (417 nucleotides). (B) Determination of the levels of positive-strand HCV RNA present in the stable cell lines by RPA. A total of 10 μg of total cellular RNA extracted from stable cell lines was hybridized with 105 cpm of the [32P]UTP-labeled negative-strand 3′ UTR RNA probe and 4×104 cpm of human β-actin RNA probe. After RNase digestion, the RNA products were analyzed in a 6% polyacrylamide-7.7M urea gel, autoradiographed, and quantified with a phosphorimager. (C) Determination of the levels of negative-strand HCV RNA by RPA. RPA is done in the same way as described in panel B except that the HCV strand-specific RNA probe was the positive-strand 5′ UTR RNA, which is complementary to the negative-strand HCV 3′ UTR region (25).