Pmirglo firefly luciferase vector

To study the functional interaction between microRNA and its targets, fragments contain the wild-type binding site (WT) or the mutated sequence (MUT) were synthesized and cloned into PmirGLO firefly luciferase vector respectively (Promega, CA, USA). Cells were co-transfected with 0.5 µg reporter plasmid and 0.5 µg Renilla luciferase (hRlucneo) control plasmid in the presence of miR-421 mimic or miR-NC in a 96-well plate (5000 cells/well) using Lipofectamine 3000 reagent according to the manufacturer’s instructions. After 48 hours, the relative luciferase activities were determined using Dual-Luciferase Reporter Assay Kit (Promega) on a luminescence microplate reader. The relative firefly luciferase activity in the reporter was normalized to that of Renilla luciferase control plasmid.

To demonstrate the functional interaction between miR-27a-3p and NOVA1 mRNA, the sequence containing the wild-type binding site in 3'UTR of NOVA1 mRNA or the sequence with mutated binding site were cloned into the PmirGLO firefly luciferase vector (Promega, CA, USA). The reporter plasmid and Renilla luciferase (hRlucneo) control plasmid were co-transfected into cells in the presence of miR-27a-3p mimic or miR-NC using Lipofectamine 2000 reagent. 48 h after transfection the relative luciferase activities were measured using Dual-Luciferase Reporter Assay Kit (Promega, CA, USA) on a microplate reader.