Pmir report luciferase wt vector

Wild-type (WT) and mutant (MT) seed region sites of hsa-miR-1246 containing 3’UTR sequence of target genes were constructed and cloned into pMIR-REPORT-Luciferase vector (Invitrogen, Carlsbad, CA, USA) (Figure 3B). Constructed insert sequences were verified by Sanger sequencing using BigDye Terminator v3.1 kit (Applied Biosystems, Foster City, CA, USA) and 3500 Series Genetic Analyzer (Applied Biosystems, Foster City, CA, USA). A previously well-established workflow for dual luciferase assay using AGS cells was applied for this experiment [57 (link),58 (link)]. Cells were co-transfected with hsa-miR-1246 mimic and miR-Control, pMIR-REPORT-Luciferase-WT vector, pMIR-REPORT-Luciferase-MT vector, and pMIR-REPORT-ß-galactosidase control vector (Invitrogen, Carlsbad, CA, USA). Luciferase activity was detected after 48 h of incubation by the Dual-Light™ Luciferase & β-Galactosidase Reporter Gene Assay System (Invitrogen, Carlsbad, CA, USA) and normalized with ß-galactosidase activity using Tecan Genios Pro (Tecan, Männedorf, Switzerland). At least three independent experiments were performed.