RNA Immunoprecipitation Assay

RNA immunoprecipitation (RIP) was performed using the EZ-Magna RIP RNA-Binding Protein Immunoprecipitation Kit (Millipore, Billerica, MA, http://www.emdmillipore.com/) according to the manufacturer’s protocol [⁴²]. The direct binding of miRNA-mediated RNA-induced silencing complex (RISC) to HCV RNA was determined using anti-Ago2 antibody (Abcam, Cambridge, MA, http://www.abcam.com/) mediated RNA pulldown followed by quantitative polymerase chain reaction (qPCR) examine the amount of HCV RNA. In brief, 70%–80% confluence cells were washed twice with PBS and trypsinized. Cells were lysed and centrifuged at 12,000 rpm for 10 minutes at 4°C. Cell lysate was incubated with antibody/beads for 18 hours at 4°C. After incubation, beads were washed three times in NT2 buffer (50 mM Tris-HCl, 300 mM NaCl, 1 mM MgCl₂, 0.05% Nonidet P-40, RNase inhibitor), followed by treated with DNase I for 15 minutes at 37°C. RNAs were extracted by RNA purification kit (RNeasy MiniElute kit, Qiagen, Hilden, Germany, https://www.qiagen.com) and cDNA was synthesized using the SuperScript kit (Thermo Fisher Scientific Life Sciences) and the quality of cDNA was analyzed by qPCR.