The regions of AATBC and CASK-3ʹ-UTR containing the wild-type (Wt) or mutant (Mut) miR-1245b-5p binding site were chemically generated by Genechem (Shanghai, China). And then they were cloned into the pmirGLO luciferase reporter gene (Promega, Madison, WI, USA), and named as AATBC-Wt/Mut and CASK-Wt/Mut. Cells were co-transfected with the indicated plasmids and miR-1245b-5p mimics using Lipofectamine 3000 reagent (Invitrogen). Luciferase activity was appraised with a Dual-Luciferase Reporter System (Promega, Madison, WI, USA).
Pmirglo luciferase reporter gene
Manufactured by Promega
Sourced in United States
The PmirGLO Luciferase Reporter Gene is a plasmid vector designed for the expression and evaluation of miRNA (microRNA) activity. The vector contains a firefly luciferase gene that can be used to measure the regulatory effects of miRNAs on gene expression.
Automatically generated - may contain errors
3 protocols using pmirglo luciferase reporter gene
1
Validating miR-1245b-5p Binding Sites
2
Validating miR-625 Binding Sites in CCND1 and LINC00511
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The 3′-UTR fragments of CCND1 containing the wild-type (Wt) or mutant (Mut) miR-625–binding site were produced by Shanghai GenePharma Co., Ltd., cloned into the pmirGLO luciferase reporter gene (Promega, Madison, WI, USA), and named as CCND1-Wt and CCND1-Mut, respectively. The luciferase plasmids, including LINC00511-Wt and LINC00511-Mut, were chemically generated in the same way as described above.
Cells were seeded in 24-well plates and cotransfected with the Wt or Mut luciferase reporter plasmid in the presence of the miR-625 mimics or miR-NC, using Lipofectamine 2000 according to the manufacturer’s protocol. Transfected cells were incubated at 37°C and 5% CO2, and harvested at 48 h after transfection. Luciferase activity was measured via a Dual-Luciferase Reporter System (Promega, Madison, WI, USA) following the manufacturer’s instructions. Firefly luciferase activity was normalized to Renilla luciferase activity.
Cells were seeded in 24-well plates and cotransfected with the Wt or Mut luciferase reporter plasmid in the presence of the miR-625 mimics or miR-NC, using Lipofectamine 2000 according to the manufacturer’s protocol. Transfected cells were incubated at 37°C and 5% CO2, and harvested at 48 h after transfection. Luciferase activity was measured via a Dual-Luciferase Reporter System (Promega, Madison, WI, USA) following the manufacturer’s instructions. Firefly luciferase activity was normalized to Renilla luciferase activity.
3
Validating miR-761 Binding to FGFR1 3'-UTR
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The putative targets of miR-761 were predicted using TargetScan v7.1 (www.targetscan.org www.microrna.org
The human FGFR1 3′-UTR region containing the wild-type (WT) or mutant (MUT) miR-761 binding site was amplified by Shanghai GenePharma Co Ltd, cloned into a pmirGLO luciferase reporter gene (Promega, Madison, WI, USA) and referred as pmirGLO-WT-FGFR1-3′-UTR and pmirGLO-MUT-FGFR1-3′-UTR, respectively. For reporter assay, cells were seeded in a 24-well plate and co-transfected with either pmirGLO-WT-FGFR1-3′-UTR or pmirGLO-MUT-FGFR1-3′-UTR, and treated with agomir-761 or agomir-NC using Lipofectamine 2000. Luciferase activities were measured using dual-luciferase reporter assay (Promega, Madison, WI, USA) at 48 h post-transfection following the manufacturer’s instruction. Firefly luciferase activity was standardized to Renilla luciferase activity.
The human FGFR1 3′-UTR region containing the wild-type (WT) or mutant (MUT) miR-761 binding site was amplified by Shanghai GenePharma Co Ltd, cloned into a pmirGLO luciferase reporter gene (Promega, Madison, WI, USA) and referred as pmirGLO-WT-FGFR1-3′-UTR and pmirGLO-MUT-FGFR1-3′-UTR, respectively. For reporter assay, cells were seeded in a 24-well plate and co-transfected with either pmirGLO-WT-FGFR1-3′-UTR or pmirGLO-MUT-FGFR1-3′-UTR, and treated with agomir-761 or agomir-NC using Lipofectamine 2000. Luciferase activities were measured using dual-luciferase reporter assay (Promega, Madison, WI, USA) at 48 h post-transfection following the manufacturer’s instruction. Firefly luciferase activity was standardized to Renilla luciferase activity.
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