The EGR3 wild-type (Wt) and mutant (Mt) 3′-UTR sequences were synthesized and sub-cloned into the psiCHECK luciferase reporter plasmid (Promega, Madison, WI, USA). 5-8F was seeded into a 12-well plate at a density of 5 × 106 and each recombinant luciferase reporter plasmid and the miR-483-5p mimic were co-transfected using Lipofectamine 2000 reagent (Invitrogen) according to the manufacturer’s instructions. The psiCHECK promoter vector was used as the control, and the pRL-SV40 Renilla luciferase vector (Promega) was used to normalize the activity of firefly luciferase. Twenty-four hours later, the luciferase activity in each well was detected using the Dual-Luciferase Reporter Assay System (Promega).
Psicheck luciferase reporter plasmid
Manufactured by Promega
Sourced in United States
The PsiCHECK luciferase reporter plasmid is a dual-luciferase reporter system designed for convenient and quantitative analysis of gene expression in mammalian cells. It contains a Renilla luciferase gene as the primary reporter and a Firefly luciferase gene as the internal control for normalization.
Automatically generated - may contain errors
2 protocols using psicheck luciferase reporter plasmid
1
EGR3 3'UTR Luciferase Assay
2
Plasmid Construction and miRNA Transfection
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To construct the plasmids expressing miR-30a-3p or miR-30-5p, the fragments of pri-miR-30a were amplified using PCR, and then, respectively, cloned into the lentiviral vector pLVTHM (Addgene Inc., Cambridge, MA, United States). The mimics, negative controls, and inhibitors of miR-30a-3p or miR-30a-5p were purchased from Genecopoeia (Guangzhou, Guangdong, China), and transfected into cells with Lipofectamine 2000 reagent (Invitrogene) according to the manufacturer’s instructions. To perform luciferase assay, small regions containing the target sequences of miR-30a-3p or miR-30a-5p in 3’-UTR were generated by PCR amplification, and cloned into psi-CHECK luciferase reporter plasmid (Promega). Two concentrations of miR-30a-3p or miR-30a-5p-mimics (10 nmol/L and 20 nmol/L) plus wild-type or mutant 3’-UTR of the target genes were applied. The primers used are listed in Table S2.
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