Viral nucleic acid extraction kit

The expression vector (pET102/D-TOPO), Ni-NTA purification system, anti-His Tag antibody, and HRP-conjugated anti human against IgG were purchased (Invitrogen, Frankfurt, Germany). Primers were synthesized by Metabion (Frankfurt, Germany). Micro bicinchoninic acid (BCA) protein assay Kit (Pierce, Rockford, United States) and Pfu DNA polymerase (Stratagene, La Jolla, United States) were provided. Viral Nucleic acid extraction Kit, PCR product purification Kit, and Plasmid extraction Kit all were obtained from Roche (Frankfurt, Germany). The Escherichia coli BL21 (DE3) strain was provided by virology laboratory of Iranian Blood Transfusion Organization. Polyvinylidene difluoride (PVDF) membrane and horseradish peroxidase (HRP) substrate for western blotting were obtained from Amersham (London, United Kingdom).

Viral RNA was extracted from the plasma of an Iranian patient with HCV subtype 1a, according to the manual of the viral nucleic acid extraction kit (Roche, Germany). cDNA synthesis was performed on 10 μL of the extracted RNA with 4 μL mixture of reverse transcription (RT) buffer, 1 μL RNase inhibitor, 1 μL dNTP mixture (10 mM), 1 μL random hexamer primer, and 1 μL MMLV RT enzyme (Thermo Scientific, CA). The reaction was carried out at 42°C for 60 minutes and 72°C for 15 minutes in a final step for inactivation of RT enzyme.