Pmirglo dual luciferase mirna target expression vector

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.

Oligos specific to the wildtype (WT) PART1 miRNA 937-5p binding region and the mutated version of the sequence (MUT) are listed in Table S2. To make double stranded sequences for cloning, the oligos were admixed into oligo annealing buffer and heated to 90 °C for 3 min, followed by cooling to 37 °C for 15 min. The WT and MUT annealed oligos (ThermoFisher Scientific) were cloned into the multiple cloning site of the pmirGLO Dual-Luciferase miRNA Target Expression Vector (ThermoFisher Scientific, using SacI and XhoI restriction enzymes (New England Biolabs Ltd.). The confirmed vectors were co-transfected into HCC1806 cells with the pRLTK vector (Promega ThermoFisher Scientific), using TransIT-BRCA transfection reagent. 24 h later the mirVana miRNA negative control mimic or mimic-hsa-miR-937-5p (ThermoFisher Scientific) was transfected into the cells using TransIT-BRCA. The resulting firefly and renilla luciferase activity in the cells were measured 24 h later using the Dual-Glo^® Luciferase Assay System (ThermoFisher Scientifc) with a SpectraMax^® M3 Multi-Mode Microplate Reader (ThermoFisher Scientific). Binding of the mimic sequence to the luciferase reporter vector would inhibit production of luminescence.