Detection of positive- and negative-strand HCV-RNA in infected cell cultures via RT-PCR. Posititve strands were assayed using the cell culture supernatant while the negative strands were assayed using total RNA purified from the cells. (A) Determining the optimum day to harvest HCV for RNA extraction and analysis. Approximately one million cells of culture #081 were divided into seven flasks and incubated. One flask was harvested on each of the following days and assayed for positive and negative strand RNA. (B) Quantitation of molecules of positive-strand HCV-RNA per ml of cell culture supernatant via real-time RT-PCR. (C) Positive- and negative-strand HCV-RNA in different cells infected with CIMM-HCV. Lane 1 CIMM-HCV, lane 2 T-cells (112 A), lane 3 B-cells (112 B), lane 4 non-committed lymphoid cells (112 AB), lane 5 the 4th serial transmission into immortalized cord B-cells (PCLB T4), lane 6 T-cells (200 A), lane 7 B-cells (200 B), lane 8 uninfected B-cells, lane 9 HCV infected patient serum, and lane 10 negative PCR control.