Mutational analysis of the miR-30-luc precursor. (A) Design of miR-30-luc. Sequences underlined are derived from the firefly luciferase mRNA, with the bottom strand being antisense to the mRNA. Single point mutations are indicated. (B) Expression of mature miR-30-luc variants as determined by primer extension. −, Cells transfected with pCMV/miR-21-luc (lane 1); +, 20 pg of an oligonucleotide encoding the intended miR-30-luc, the underlined bottom strand in A (lane 3). Arrow indicates the expected position of miR-30-luc. (C) Effect of miR-30-luc on luciferase expression. 293T cells were transfected with pCMV/luc, pRL-CMV, and plasmids encoding a wild-type or mutant miR-30-luc precursor. A dual luciferase assay was performed 2 d later. The ratio of firefly luciferase to Renilla luciferase observed in cells cotransfected with the parental pBC12/CMV vector was set at 1 (shown by −).
