Prl cmv renilla luciferase reporter

The 293T cells were seeded in 96-well plates and co-transfected with either pRL-CMV Renilla luciferase reporter, firefly luciferase reporter, or miR-491 mimic. The luciferase activities of Renilla and firefly reporter vectors were quantified using a dual-luciferase reporter assay according to the manufacturer's protocol (Promega Corporation) after 48 h of incubation. The 3′ untranslated regions (UTRs) were taken from FOXP4, and the binding site of circ-0000212 with miR-491 was taken from circ-0000212. The sequences containing the miR-491-binding sites or point mutations in the binding sites were inserted between SpeI and HindIII sites in the firefly luciferase reporter vectors, in order to generate wild-type and mutant luciferase reporter vectors, respectively. These sequences were all obtained from Shanghai GenePharma Co., Ltd. The pRL-CMV Renilla luciferase reporter was purchased from Promega Corporation. Lipofectamine 2000 was used to perform transfection according to the manufacturer's protocol. Briefly, the 293T cells were seeded 24 h prior to transfection at 50–60% confluence. The cells were transfected once they reached ~70% confluence with 50 ng plasmids, and 20 nM miRNA mimics or miRNA inhibitor, according to the manufacturer's protocol. Luciferase activity was measured 48 h post-transfection and Renilla luciferase activity was used as a control.