We used miRWalk (http://mirwalk.umm.uni-heidelberg.de/ ), miRMap (https://mirmap.ezlab.org/ ) and TargetScan (http://www.targetscan.org/ ) to predict the upstream miRNAs of VCL. A dual luciferase reporter assay was performed to prove the regulation of miR-663b and VCL in 293 T cells. Wild-type (WT) and mutant (MUT) VCL 3′-UTRs were synthesized and inserted into the pmiR-REPORT luciferase plasmid (OBIO, Shanghai, China). Then, pmiR-REPORT Luciferase-VCL 3′UTR (WT), pmiR-REPORT Luciferase-VCL 3′UTR (MUT) and pRL-CMV (CON) (Promega, WI, USA) were cotransfected into 293T cells with miR-663b mimics or NC (GenePharma, Shanghai, China). After 48 h of transfection in a 96-well plate, a dual fluorescence luciferase reporter gene detection system (Promega, WI, USA) was used to detect fluorescence. The experiments were normalized to Renilla luciferase activity.
Dual fluorescence luciferase reporter gene detection system
Manufactured by Promega
Sourced in United States
The Dual fluorescence luciferase reporter gene detection system is a lab equipment product that enables the simultaneous detection of two different luciferase reporter genes. It provides a platform for measuring the activity of two distinct reporter genes within the same sample.
Automatically generated - may contain errors
Lab products found in correlation
2 protocols using dual fluorescence luciferase reporter gene detection system
1
Validating miR-663b Regulation of VCL
2
Validating miR-663b Target Genes
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PicTar (https://pictar.mdc-berlin.de/ ), TargetScan (http://www.targetscan.org/ ) and miRanda (http://www.microrna.org/microrna/home.do ) were used to predict miR-663b targets. The expression of the miR-663b-target gene was verified in 293T cells by dual luciferase reporter gene assay. First, we insert the 3′-UTR of wild-type (WT) and mutant (MUT) MGAT3 into the psiCHECKTM-2 plasmid (Promega Corp.). Then, psiCHECKTM-2-MGAT3-3′-UTR-WT or psiCHECKTM-2-MGAT3-3′-UTR-MUT were co-transfected into 293T cells with miR-663b mimics or its NC. After 48 h of transfection in a 96-well plate, a Dual Fluorescence Luciferase reporter gene detection system (Promega Corp.) was used to detect the fluorescence.
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