The biological prediction website microRNA.org was used to analyze target genes of miR-379 and also to verify whether EIF4G2 was a direct target gene of miR-379. After cloning and amplifying the full length of EIF4G2 in 3′-UTR region, the PCR products were cloned into multiple cloning sites of pmirGLO luciferase (Promega Corp., Madison, WI, U.S.A.), which was named pEIF4G2-Wt. Site-specific mutagenesis was performed to alter the binding site of miR-379, which was predicted by bioinformatics, followed by construction of the pEIF4G2-Wt vector. The number of cells and transfection efficiency were normalized using pRL-TK (TaKaRa Biotechnology Ltd., Dalian, China) as an internal reference, which expressed Renilla luciferase. miR-379 mimics and miR-379 mimic NC were respectively cotransfected with the luciferase reporter vector into U2OS and MG-63 cells, followed by dual luciferase activity detection according to the instructions given by Promega. The experiment in each group was repeated three times. The comparison among the groups was analyzed by t test.
Dual luciferase activity detection
Manufactured by Promega
Sourced in United States
The Dual Luciferase Activity Detection system is a laboratory instrument that measures luminescence from two different luciferase reporter enzymes within the same sample. It provides a quantitative analysis of the activities of these two reporter enzymes, which can be used to study gene expression and other biological processes.
Automatically generated - may contain errors
3 protocols using dual luciferase activity detection
1
Validating miR-379 Targeting of EIF4G2
2
Investigating SNP Effects on miRNA Interactions
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Reporter plasmids harboring SNP sites at rs1804029 and rs17848060 loci in CXCR4 were synthesized from Shanghai Integrated Biotech Solutions (Shanghai, China) and were named as pGL3‐CXCR4‐rs1804029 T, pGL3‐CXCR4‐rs1804029 G, pGL3‐CXCR4‐rs17848060 T, and pGL3‐CXCR4‐rs17848060 A. To investigate the effect of SNP on miRNA and target sequence interaction, pGL3‐CXCR4‐rs1804029 T or pGL3‐CXCR4‐rs1804029 G was co‐transfected with hsa‐miR‐146a‐3p mimic or hsa‐miR‐146a‐3p inhibitor, and pGL3‐CXCR4‐rs17848060 T or pGL3‐CXCR4‐rs17848060 A was co‐transfected with hsa‐miR‐20a‐3p mimic or hsa‐miR‐20a‐3p inhibitor, respectively, into HEK293T cells using Lipofectamine 2000 (Invitrogen, Carlsbad, CA, USA), and cells were harvested at 24 hours post‐transfection for dual‐luciferase activity detection according to commercial manual instructions (Promega, Madison, WI, USA).
3
CXCR4 SNP Impact on miRNA Interaction
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Reporter plasmids harboring SNP sites at rs1804029 and rs17848060 loci in CXCR4 were synthesized from Shanghai Integrated Biotech Solutions (Shanghai, China) and were named as pGL3-CXCR4-rs1804029 T, pGL3-CXCR4-rs1804029 G, pGL3-CXCR4-rs17848060 T, and pGL3-CXCR4-rs17848060 A. To investigate the effect of SNP on miRNA and target sequence interaction, pGL3-CXCR4-rs1804029 T or pGL3-CXCR4-rs1804029 G was co-transfected with hsa-miR-146a-3p mimic or hsa-miR-146a-3p inhibitor, and pGL3-CXCR4-rs17848060 T or pGL3-CXCR4-rs17848060 A was co-transfected with hsa-miR-20a-3p mimic or hsa-miR-20a-3p inhibitor, respectively, into HEK293T cells using Lipofectamine 2000 (Invitrogen, Carlsbad, CA, USA), and cells were harvested at 24 h post transfection for dual-luciferase activity detection according to commercial manual instructions (Promega, Madison, WI, USA).
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