Pmir rb report dual luciferase reporter

The binding sites between miR-195-5p and Bcl-2 were predicted using TargetScan bioinformatics software (version 7.1; www.targetscan.org/vert_71). To confirm predictions, dual luciferase reporter assays were performed. The wild-type (WT)-Bcl-2 and mutant (MUT)-Bcl-2 3′-UTRs of Bcl-2 were cloned into a pmiR-RB-Report™ dual luciferase reporter gene plasmid vector (Guangzhou RiboBio Co., Ltd.) according to the manufacturer's instructions. HUVECs were co-transfected with WT-Bcl-2 or MUT-Bcl-2 and miR-195-5p mimic or mimic control by using Lipofectamine^® 2000 (Invitrogen; Thermo Fisher Scientific, Inc.) at 37°C for 48 h. A total of 48 h after cell transfection, the luciferase activity was analyzed using the dual-luciferase assay system (Promega Corporation). Luciferase activity was normalized to the Renilla luciferase activity in the current study.

Wild-type (WT-BAK1) and mutant (MUT-BAK1) 3'UTR BAK1 were cloned into pmiR-RB-Report™ dual luciferase reporter gene plasmid vectors (Guangzhou RiboBio Co., Ltd.), according to the manufacturer's protocol. 293T cells (5x10⁴ cells per well; American Type Culture Collection) were co-transfected with WT-BAK1 or MUT-BAK1, and miR-345-3p mimic or mimic control using Lipofectamine^® 2000 (Invitrogen; Thermo Fisher Scientific, Inc.) according to the manufacturer's protocol. Following incubation at 37̊C for 48 h, luciferase activities were detected using a Dual-luciferase assay system (Promega Corporation), according to the manufacturer's protocol. Firefly luciferase activity was normalized to Renilla luciferase activity.