Gel shift assays to analyze hDicer:pre-miRNA interaction. (A) Increasing amounts of hDicer (0, 0.1, 0.4, 1, 3, 7, and 20 nM) were incubated with ∼0.3 nM 32P-labeled pre-let-7a or pre-miR-412 on ice for 40 min, before fractionation on a 7.5% native gel. (B) Increasing amounts of hDicer were incubated with the indicated pre-miRNAs. (C) hDicer was incubated (“+”) with the indicated pre-miRNAs before gel electrophoresis. (D) Comparisons of the relative hDicer cleavage efficiencies and binding efficiencies for the pre-miRNAs listed in C. Averaged percentages from at least two replicate tests are shown. For hDicer cleavage efficiencies, see Supplemental Table 1. Relative hDicer binding efficiency of a pre-miRNA was calculated by normalizing its binding to that of pre-let-7a, which was set at 100%.
