Luciferase Reporter Assay for miRNA Targeting

The HEK-293T cell line (American Type culture collection, Manassas, VA) was cultured in DMEM. When achieving 80–90% confluence, cells were detached by 0.25% trypsin, passaged and cultured at 37 ºC with 5% CO₂. Logarithmically growing cells were used for further experiments. The artificially synthesized TGIF1 3’untranslated region (3’UTR) was introduced into pGL3-control (Promega Corp., Madison, WI) through endonuclease sites XhoI and BamH I. The mutation site of complementary sequence of seed sequence was designed on TGIF1-wild-type (WT). After restriction enzyme digestion, the target fragment was inserted into pGL3-control vector using T4 DNA ligase. The correctly sequenced luciferase reporter plasmids WT and mutant type (MUT) were co-transfected with miR-494 mimic/mimic-NC into HEK-293T cells, respectively, for 48 h. The luciferase activity was tested utilizing Dual-Luciferase Reporter Assay System Kit (Promega) on a TD-20/20 luminometer (E5311, Promega) [30 (link)].