Dual luciferase detection system kit

Luciferase reporter vectors (circNINL-WT, circNINL-MUT, FGFR1 3'UTR-WT and FGFR1 3'UTR-MUT) were generated by inserting circNINL or FGFR1 3'UTR sequence containing miR-3918 target site or mutated site into pGL3 vector (Promega). The vectors were co-transfected into A549 cells with miR-3918 mimic by Lipofectamine® 3000, and after 48 h, cell luciferase activity was checked by dual luciferase detection system kit (Promega) [28 (link)].

After sequence comparison between circ_0058106 (or Wnt2b) and miR-185-3p, the fluorescein-labeled reporter gene was detected using the dual-luciferase detection system kit (Promega, Madison, USA). Fadu cells were seeded on a 48-well plate, cultured for 24 h, then co-transfected with wild-type or mutant pmiRGlo-circ_0058106 (or pmiRGlo-Wnt2b) dual-luciferase reporter vectors incorporating miR-185-3p binding sites vectors and miR-185-3p mimics or negative control by using transfection reagents. After 48 h, added passive lysis buffer (20 μL) and Luciferase Assay Reagent II (100 μL) to each well, measured the absorbance at 580 nm using a microplate reader. Then added Stop&Glo^® Reagent (100 μL) to the same well and measure the absorbance at 460 nm.