FIGURE 2.

miR-30 mediated repression of Pol II-driven reporter constructs. (A) Raw RLU values for the wild-type and mutant HCV IRES Pol II constructs. (B) Expression of miR-21 (upper panel) and miR-30 (lower panel) was evaluated by primer extension analysis. miR-21 or miR-30 expression plasmids were transfected into 293T cells, and primer extension was performed on RNAs harvested 24 h later. No miRNA was expressed in mock-transfected samples. (C) 293T cells were co-transfected with miR-21 or miR-30 plasmids and the indicated reporter construct. Cells were harvested for analysis at the indicated time points. (Darker shaded bars) Time points used for characterization of target mRNA integrity. A plasmid encoding FLuc was used as a transfection control. (D) Quantitative RT-PCR (qRT-PCR; upper panel) for RLuc mRNA was normalized to endogenous GAPDH mRNA levels. Target mRNAs co-transfected with miR-21 are set to 100%. Northern blots (lower panel) for RLuc mRNAs were performed as described in the Materials and Methods. rRNAs from corresponding samples are shown for loading control. qRT-PCR experiments were repeated on at least three separate occasions.