Additional file 2.

Dicer in functionally competent in Huh-7 and 9–13 cells. The data provided indicate that the activity of Dicer is not influenced by HCV in vivo. The human pre-let7a-3 RNA was transcribed and randomly labeled (α-³²P UTP, Perkin Elmer) by in vitro transcription using T7 promoter (Ambion) and purified by 10% denaturating PAGE. Huh-7 and 9–13 cells were resuspended in lysis buffer (Tris·HCl 50 mM, 137 mM NaCl, Triton X-100 1%) and immunoprecipitation (IP) was performed on 1 mg of proteins incubated with protein-G beads alone or beads/rabbit anti-Dicer at 4°C for 3 hours. Immune complexes were washed 3 times in lysis buffer, following by an additional wash in Tris·HCl 20 mM and MgCl₂ 2 mM, pH 7.5. α-³²P labeled pre-let7a-3 RNA was incubated with immune complexes for in vitro processing of pre-miRNA in Dicer RNase activity assay for 1 hour at 37°C in Tris·HCl 20 mM, DTT 1 mM, ATP 1 mM, MgCl₂ 5 mM and 5% SUPERase∙In (Ambion), pH 7.5. Proteins were extracted by phenol/chloroform and RNA was precipitated and analyzed by denaturating PAGE and autoradiography.

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Ouellet et al. Journal of Negative Results in BioMedicine 2009 8:8   doi:10.1186/1477-5751-8-8