A dual-luciferase reporter assay was performed to observe the binding between miR-31-5p and BAP1. According to the operating instructions of the dual-luciferase reporter gene detection kit (Beyotime, China), the target gene and internal reference gene plasmids labeled with luciferase were grouped and co-transfected with H9C2 cells for 48 h. The ratio of plasmids carrying the target gene to those carrying internal references was 50:1. PmirGLO-BAP1 3-’ UTR-WT and PmirGLO-BAP1 3-UTR-mut were used as controls. Luciferase expression was measured using a JP-K6000 chemiluminescence analyzer (Jiapeng, China).
Dual luciferase reporter gene detection kit
Manufactured by Beyotime
Sourced in China
The Dual-luciferase reporter gene detection kit is a laboratory tool designed for the analysis of gene expression. It utilizes two different luciferase reporter enzymes, Firefly luciferase and Renilla luciferase, to enable quantitative measurement of transcriptional activity.
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Lab products found in correlation
Lipofectamine rnaimax, by Thermo Fisher Scientific (1 mentions)
293t cell, by American Type Culture Collection (1 mentions)
Lipofectamine 2000 reagent, by Thermo Fisher Scientific (1 mentions)
Anti actin antibody, by Abcam (1 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (1 mentions)
Trizol, by Thermo Fisher Scientific (1 mentions)
Opti mem, by Thermo Fisher Scientific (1 mentions)
Runx2 antibody, by Abcam (1 mentions)
Alp detection kit, by Beyotime (1 mentions)
Anti lrp6 antibody, by Abcam (1 mentions)
Psicheck 2 luciferase reporter vector, by Promega (1 mentions)
Multiplate reader, by Agilent Technologies (1 mentions)
Pgl3 vector, by Promega (1 mentions)
Lipofectamine 2000, by Thermo Fisher Scientific (1 mentions)
Luciferase reporter system, by Promega (1 mentions)
Psicheck 2 plasmid, by Promega (1 mentions)
Quickmutation kit, by Beyotime (1 mentions)
14 protocols using dual luciferase reporter gene detection kit
1
Dual-Luciferase Assay for miR-31-5p and BAP1
2
Dual-Luciferase Assay for miRNA-Target Interaction
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The dual-luciferase reporter plasmid was purchased from Han Bio (Han Bio, Shanghai, China). HEK-293T cells were cultured in 6-well plates at a density of 1 × 105 cells/well. Cells were co-transfected with a plasmid mixture with or without the mir-23b-5p 3′-UTR (500 ng) and TMEM127 3′-UTR or negative control (NC) (10 nM final concentration) using Lipofectamine RNAiMAX (Invitrogen). Luciferase activity was measured after 48 h with a dual-luciferase reporter gene detection kit (Beyotime, Shanghai, China).
3
Dual Luciferase Assay for Smad7 Overexpression
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The dual luciferase reporter gene detection kit (Beyotime, China) was used to evaluate the firefly and Renilla luciferase activity 24 hours after transfection. Then, we use Lipofectamine 3000 reagent to transfect MHCC97H and HepG2 cells with Smad7 overexpression plasmid and control vector in a 24-well plate.
4
Luciferase Assay for miRNA-lncRNA Interaction
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The 293T cells (ATCC, Manassas, USA) were inoculated into 96-well culture plates. When the cells fusion degree reached 60%, miR-193b-3p mimic or mimic NC was cotransfected into cells with IGFBP5 and lnc-5423.6 wild-type and mutant double luciferase reporter vectors using Lipofectamine 2000 reagent (Invitrogen). After 48 h, the firefly luciferase and Renilla luminescence luciferase activities were detected by a dual luciferase reporter gene detection kit (Beyotime, Shanghai, China) and a multifunctional enzyme standard (Thermo Fisher Scientific).
5
Osteogenic Differentiation of BMSCs
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Primary cells of Human Bone Marrow-Derived Mesenchymal Stem Cells (BMSCs) (Shanghai Zhong Qiao Xin Zhou Biotechnology Co., Ltd.); DMEM medium, FBS (Gibco, USA); ALP detection kit (Beyotime Biotechnology Co., Ltd.); Trizol (Thermo Fisher Scientific Company, USA); Anti-BMP antibody, anti-OPG antibody, RUNX2 antibody, anti-GSK-3 antibody, anti-Frizzled 1 antibody, anti-Lrp5 antibody, anti-Lrp 6 antibody, and anti-actin antibody (Abcam Company, USA); 293T cells (Sciarray Biotech Co., Ltd.); hsa-miR-31 mimics, hsa-miR-31 mimics NC, hsa-miR-31 inhibitor, and hsa-miR-31 inhibitor NC (Shanghai Genepharma Co., Ltd.); Lipossome 3000 transfection reagent (Thermo, USA); Serum-free culture medium, Opti-MEM (Invitrogen, USA); Dual luciferase reporter gene detection kit, Cat #: RG009 (Beyotime Biotechnology Co., Ltd).
6
Validating circMaml2-miR-683-Sec62 Interaction
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The bioinformatic websites miRWalk and TargetScan were used to predict the binding between circMaml2 and miR-683 and between miR-683 and Sec62. The results were verified by performing a dual-luciferase reporter gene experiment according to the predicted binding sites obtained from the bioinformatic websites miRanda and TargetScan. Wild-type and mutant circMaml2 transcripts (NR_033241.1) and Sec62 3′-UTR were introduced into the psiCHECK-2 luciferase reporter vector (Promega, Madison, WI, USA) and co-transfected with the miR-683 mimic or NC into MC38 cells. Finally, the relative luciferase activity was assessed by the dual-luciferase reporter gene detection kit (Beyotime, Shanghai, China).
7
Luciferase Reporter Assay for miRNA Targets
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According to the information on the binding site of Gm16062 and Mef2a-3′UTR with the miR-129-5p seed sequence, respectively, pmirGLO-Gm16062-WT (Gm16062-WT), pmirGLO-Gm16062-Mutate (Gm16062-MUT), pmirGLO-Mef2a-3′UTR-WT (Mef2a-WT), and pmirGLO-Mef2a-3′UTR-Mutate (Mef2a-MUT) were manufactured by TsingKe Biotech, respectively. For luciferase reporter analysis, plasmid or nucleic acid molecules were transfected into the cells according to the experimental design, using Lipofectamine3000 or HiPerFect. After 48 h, the luciferase activity analysis was performed using the Dual Luciferase Reporter Gene Detection Kit (Beyotime, Shanghai, China). Firefly luciferase activity was normalized against Renilla luciferase activity.
8
Nrf2 Promoter Activity Assay
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The dual luciferase reporter assay was performed in Caco-2 cells that had been transfected with shRNA and treated with Tg treatment. The cell culture medium was replaced by a serum-free medium when the density reached 70%. The pGL4.37 (luc2P/ARE/Hygro) plasmid was transfected for 12 h, and the serum-free medium was converted to a complete medium. After 24 h of transfection, the detection preparation was completed as per the instructions stipulated in the dual-luciferase reporter gene detection kit (#RG027, Beyotime, China). The fluorescence intensity was measured by a multi-functional enzyme labeling instrument, and the activity of the Nrf2 promoter was calculated.
9
Characterization of circRALGPS2 RNA Structure
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According to the RNA secondary structure of circRALGPS2, different plasmids were constructed using the Luc2-IRES-Reporter vector (Geneseed). The Luc2-IRES-Reporter vector plasmid and dual-luciferase reporter gene were constructed and synthesized by Beijing Tsingke Biotech Co., Ltd. Chicken DF1 cells were then collected and transfected with the plasmids. Next, luciferase activity was detected using a dual-luciferase reporter gene detection kit (Beyotime, Shanghai, China). The luminescent activities of firefly luciferase and Renilla luciferase were finally detected using a multi-plate reader (Biotek, Winooski, VT, USA), according to the manufacturer’s instructions.
10
Investigating TCF4 and β-catenin Binding Sites
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The putative binding sites with TCF4 or β-catenin in the MFF promoter were amplified and cloned into the pGL3 vectors (Promega, Madison, WI, USA) using partial sequences. Subsequently, pGL3 vectors were co-transfected into HK-2 cells with TCF4 or β-catenin overexpression vector. Following a 48 h transfection period, cellular lysis was performed and luciferase activity was measured using a microplate reader following the guidelines provided by the dual-luciferase reporter gene detection kit (Beyotime, Shanghai, China).
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