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. Author manuscript; available in PMC: 2015 May 6.
Published in final edited form as: Antiviral Res. 2010 Jul 14;88(1):119–123. doi: 10.1016/j.antiviral.2010.07.004

Figure 2. Distribution of miR-122 in equilibrium density gradients.

Figure 2

P16 fractions were layered on top of a linear 20-70% sucrose gradient, and centrifuged at 35 000 rpm for 18 hrs at 4°C in an SW60 rotor. (A) Fractions were analyzed for density and total protein (left panel) and total RNA (right panel). (B) Fractions were concentrated by ultracentrifugation (TLS-55 rotor, 52 000 rpm for 2 hrs at 4°C) and increasing quantities tested for replicase activity (top panel). HCV RNA was detected in a one-step RT-PCR assay (Qiagen), using the primers: 5′-ATC CGC TTG TGG CAG AGG AG, and 5′-CCT GGA GAG TAA CTG TGG AGT (bottom panel). (C) Concentrated fractions were analyzed by immunoblot for Ago2, NS3, and NS5B proteins. (D) miR122 was detected by an RNase protection assay (mirVana miRNA Detection Kit, Applied Biosystems); protected fragments were resolved on 15% polyacrylamide/8M urea gels, and imaged by autoradiography. Undigested probe (UDP) and protected probe (PP) bands are indicated.