Complementary oligonucleotide pairs comprising a portion of putative miRNA binding sites were synthesized (Thermo Fisher Scientific), annealed and cloned into the pmirGlo Dual Luciferase miRNA target expression vector (Promega Corporation, USA) between the NheI/NotI restriction sites of the multiple cloning site downstream of a luciferase gene.
For luciferase assays, HEK293T cells were seeded into 96-well cell culture pates. 24 h later, the cells were co-transfected with 200 ng of the pmirGlo Dual Luciferase miRNA target expression vector and miR-744-5p, microRNA inhibitor anti-miR-744-5p or non-targeting siRNA control (NT) at a final concentration of 50 nM using Lipofectamine 3000 (Thermo Fisher Scientific). Three days after transfection, cells were lysed with the Dual-Glo Reagent (Dual-Glo Luciferase Assay System; Promega Corporation, Mannheim, Germany) and luciferase activity was quantified on a SpectraMax M5e microplate reader (Molecular Devices, Sunnyvale, USA). After calculating the ratio of firefly luminescence to the luminescence from Renilla, the experimental well ratio was normalized to the ratio of the control wells.
For luciferase assays, HEK293T cells were seeded into 96-well cell culture pates. 24 h later, the cells were co-transfected with 200 ng of the pmirGlo Dual Luciferase miRNA target expression vector and miR-744-5p, microRNA inhibitor anti-miR-744-5p or non-targeting siRNA control (NT) at a final concentration of 50 nM using Lipofectamine 3000 (Thermo Fisher Scientific). Three days after transfection, cells were lysed with the Dual-Glo Reagent (Dual-Glo Luciferase Assay System; Promega Corporation, Mannheim, Germany) and luciferase activity was quantified on a SpectraMax M5e microplate reader (Molecular Devices, Sunnyvale, USA). After calculating the ratio of firefly luminescence to the luminescence from Renilla, the experimental well ratio was normalized to the ratio of the control wells.