Fig. 5.

miR-US5-2 downregulates US7 using two distinct mechanisms. (A) Plasmid containing the US7 open reading frame and predicted 3′UTR or empty plasmid was transfected into cells with either negative control miRNA or the indicated miR(s). Twenty-four hours after transfection, RNA was isolated and RT-PCR analysis was performed using primer probe sets specific for US7 and GAPDH (for ΔΔCT normalization). Relative amounts of US7 mRNA are shown. (B) Reporter constructs containing the wild-type US7 3′UTR or 3′UTRs with the perfectly matched miR-US5-2 site (Mut 1), the imperfectly matched 6-mer miR-US5-2 site (Mut 2), or both (Mut 1 + 2) were mutated were transfected into cells. Twenty-four hours after transfection, RNA was isolated and RT-PCR analysis was performed using primer probe sets specific to Rluc and Fluc. Rluc expression was normalized to Fluc by ΔΔCT, and relative Rluc mRNA is shown.