TargetScan software (version 7.0; targetscan.org/ ) (18 (link)) was used to identify the miR-15b-5p binding site in the 3'-UTR of ACSS2. The wild-type (WT) and mutant (MUT) 3'-UTR sequences of ACSS2 at the miR-15b binding site were chemically synthesized by General Biosystems, Inc., and inserted into the XhoI/NotI site of psiCHECK-2 luciferase vector (Promega Corporation) to construct the recombinant plasmids psiCHECK2-ACSS2-WT and psiCHECK2-ACSS2-MUT, respectively. miR-15b-5p mimics or miR-15b-5p mimic NC were co-transfected with psiCHECK2-ACSS2-WT and psiCHECK2-ACSS2-MUT into HEK-293T cells using the Exfect Transfection reagent (Vazyme Biotech Co., Ltd.). After 48 h, firefly and Renilla luciferase activities were measured on an EnSpire reader (PerkinElmer, Inc.) using the Dual Luciferase Assay System (Dalian Meilun Biology Technology Co., Ltd.).
Dual luciferase assay system
Manufactured by Meilun
The Dual Luciferase Assay System is a laboratory equipment designed to measure the activities of two different luciferase reporter enzymes simultaneously within the same sample. It provides a reliable and efficient method for comparative gene expression analysis.
Automatically generated - may contain errors
2 protocols using dual luciferase assay system
1
Validating miR-15b-5p Regulation of ACSS2
2
Dual-Luciferase Assay of CircRNA and RBM5
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The 3′UTR of RBM5 and hsa_circ_0003176 containing wild type and mutant sequences of binding sites was cloned into pmiGLO vector (Promega, USA) to generate pmiGLO-hsa_circ_0003176 vector and pmiGLO-RBM5 vector. And then, the pmiGLO-hsa_circ_0003176 vector or pmiGLO-RBM5 vector was cotransfected to NSCLC cells with miR-182-5p mimics or NC mimics, respectively. The luciferase was detected by Dual-Luciferase Assay System (Meilunbio, MA0518) at 48 h after the transfection following the instruction of manufacturer.
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