Pmir report beta galactosidase reporter control vector

Luciferase reporter and mutagenesis assays were conducted as described previously17 (link)23 (link), with some modifications. Briefly, for transfection, HEK-293T cells were plated in 96-well plates at a concentration of 6,000 cells per well in triplicate for each condition. For the miR-22 targeting CRTC1 and FLT3 experiments, after overnight incubation, cells were transfected with 20 ng of the pMIR-REPORT bearing the CRTC1 or FLT3 3′UTR or the 3′UTRs with miR-22 binding site mutations, and 20 ng of MSCV-miR-22 or an empty MSCV vector using Effectene Transfection Reagent (Qiagen) according to the manufacturer’s protocol. pMIR-REPORT Beta-galactosidase Reporter Control Vector (Ambion) (1 ng) was co-transfected for transfection efficiency control in all transfections. Cells were lysed and firefly luciferase and β-galactosidase activities were detected using Dual-Light Combined Reporter Gene Assay System (Applied Biosystems, Foster City, CA) 48 h post transfection. Firefly luciferase activity was normalized to β-galactosidase activity for each transfected well. For the Tet1 targeting miR-22 study, HEK-293T cells were transfected with 20 ng MSCV-Tet1 construct and/or 20 ng pGL4.15-miR-22 promoter. The succeeding luciferase reporter assay was conducted according to the manufacture’s protocol (Promega). Each experiment was performed in triplicate and repeated three times.