The miRanda, RNAhybrid, and TargetScan databases were used for miR‐483‐3p target gene prediction with a consensus approach, and the intersection of the miR‐483‐3p predicted target genes and the differentially expressed proteins was calculated and shown with a Venn diagram, as previously described.20 Based on the prediction of miR‐483‐3p and Cdk9 targeting sites, the wild‐type and mutant Cdk9 target sites for miR‐483‐3p were synthesized and cloned into a pMIR‐REPORT miRNA Expression Reporter Vector System (Ambion, Austin, TX). The pMIR‐REPORT β‐gal control vector (Ambion, Austin, TX) was used as a reference. For the luciferase assay, HeLa cells were co‐transfected with wild‐type (3′UTR‐Cdk9 [wt]) plasmid with miR‐483‐3p or NC and treated with mutant (3′UTR‐Cdk9 [mut]) plasmid with miR‐483‐3p or NC. Luciferase activity was measured 48 hours after transfection using a dual‐luciferase assay kit (Promega, Madison, WI).21
Pmir report β gal control vector
Manufactured by Thermo Fisher Scientific
Sourced in United States
The PMIR-REPORT β-gal control vector is a plasmid that contains the Escherichia coli lacZ gene encoding the β-galactosidase enzyme. This vector serves as a positive control for assays involving β-galactosidase activity.
Automatically generated - may contain errors
Lab products found in correlation
Lipofectamine 2000, by Thermo Fisher Scientific (2 mentions)
Pmir report mirna expression reporter vector system, by Thermo Fisher Scientific (1 mentions)
Dual luciferase assay kit, by Promega (1 mentions)
Pmir report luciferase, by Thermo Fisher Scientific (1 mentions)
Dual light system, by Thermo Fisher Scientific (1 mentions)
2 protocols using pmir report β gal control vector
1
Validation of miR-483-3p and Cdk9 Interaction
2
Luciferase Assay for Mcl-1 3'UTR
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To construct pMIR-REPORT-Mcl-1-3′ UTR-WT and pMIR-REPORT-Mcl-1-3′ UTR-MUT plasmids, a wild type 3′ UTR segment of human Mcl-1 mRNA (1198-1725 nt, Genebank accession No. NM_021960) containing two putative miR-30 binding sites (1329–1351 and 1584–1602 nt) or a corresponding multi-base mutant sequence was cloned into the SacI and HindIII sites downstream of the firefly luciferase reporter gene in pMIR-REPORT Luciferase (Ambion, Austin, TX, USA) by BioInnovatise, Inc. (Rockville, MD). These reporters were transfected into human CD34+ cells using Lipofectamine 2000 (Thermo Fisher Scientific, Grand Island, NY, USA) and transfection efficiency was corrected by a p-MIR-report-β-gal control vector (Ambion, Austin, TX, USA). The Ambion pre-miR-30 precursors were co-transfected with pMIR-report, pMIR-hMcl-1-WT, or pMIR-hMcl-1-MUT plasmid. Luciferase and β-galactosidase activity were determined using Dual-Light System (Applied Biosystems, Bedford, MA, USA).
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