6518 Nucleic Acids Research, 2008, Vol. 36, No. 20 Figure 3. In vitro processing of hairpin RNA substrates. (A) In vitro processing assays were performed using cell extracts from wild-type or Dicer mHelicase HCT116 cells. Cell extracts (25 mg each) from either wild-type or mHelicase cells were incubated with 50-end-labeled synthetic pre-miR- 21 at 378C for 1 h, run on a 15% denaturing polyacrylamide gel and visualized by autoradiography. The Ambion Decade marker and mature miR-21 (arrowhead) are illustrated. (B) Uniformly radiolabeled RNAs corresponding to the SII-21 shRNA and SII-50 lhRNA were incubated with either the wild-type (25 mg) or mHelicase (25 mg) cell extract at 378C for 2 h and then electrophoresed in a 10% denaturing polyacrylamide gel and visualized by phosphorimager analysis. The control lane represents substrate RNA that was incubated with reaction buﬀer but no cell extract. The Ambion Decade marker is illustrated and the arrowheads mark the migration of the processed siRNAs. In the SII-50 gel, lanes 3–6 marked ‘Irrelevant samples’ were performed for a diﬀerent experiment and are not included in our analysis. The blank lane has no RNA. (C) The processing eﬃciency for each radiolabeled substrate RNA was determined by phosphorimager analysis as follows: processing eﬃciency = matureIDV/ (matureIDV + precursorIDV). IDV, Integrated Density Value. The mean and standard deviation of three experiments is represented.