Wild type (wt) and mutant (mut) 3′-UTR of STMN1 were inserted into downstream of the dual luciferase reporter vector. For luciferase assay, 105 cells were plated and cultured in 24-well plates to reach approximately 70% confluence. Cells were co-transfected with miR-101 mimic and wt/mut 3′-UTR of STMN1 dual luciferase reporter vector, respectively. After 48 hours transfection, dual luciferase reporter gene assay kit (BioVision, Milpitas, CA, USA) was used to determine the luciferase activities on luminometer (Roche, Basel, Switzerland). Renilla luciferase activity was normalized to firefly luciferase activity.
Dual luciferase reporter gene assay kit
Manufactured by Abcam
Sourced in United States
The Dual Luciferase Reporter Gene Assay Kit is a tool used to measure and compare the activities of two different reporter enzymes, typically firefly luciferase and Renilla luciferase, in a single sample. The kit provides the necessary reagents and protocols to perform this type of reporter gene analysis.
Automatically generated - may contain errors
Lab products found in correlation
Fetal bovine serum (fbs), by Thermo Fisher Scientific (2 mentions)
Lipofectamine 2000, by Thermo Fisher Scientific (1 mentions)
Plasmid extraction kit, by Promega (1 mentions)
Dual luciferase reporter assay system, by Promega (1 mentions)
Glomax 20 20 luminometer, by Promega (1 mentions)
293t cell, by American Type Culture Collection (1 mentions)
Pcdna3.1 vector, by Thermo Fisher Scientific (1 mentions)
Lipo2000, by Thermo Fisher Scientific (1 mentions)
Pgl3 basic vector, by Promega (1 mentions)
Penicillin streptomycin, by Thermo Fisher Scientific (1 mentions)
Pgl3 luciferase reporter vector, by Promega (1 mentions)
9 protocols using dual luciferase reporter gene assay kit
1
Luciferase Assay for miR-101 Target
2
Validating miR-218 Regulation of MITF
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The bioinformatics website (http://www.targetscan.org ) was adopted to predict the targeting relationship between miR-218 and MITF and binding sites of miR-218 to MITF. MITF 3′untranslated region (UTR) promoter sequence containing miR-218 binding sites were synthesized. MITF-3′UTR-wild type (WT) plasmid was constructed. According to this plasmid, binding sites were mutated to construct MITF-3′UTR-mutant type (MUT) plasmid based on the instructions of plasmid extraction kit (Promega Corporation, Madison, WI, USA). Cells at logarithmic growth phase were inoculated in the 96-well plate. When cell confluence reached 70%, transfection was performed based on the instructions of Lipofectamine 2000 (Invitrogen, Carlsbad, CA, USA). MITF-3′UTR-WT and MITF-3′UTR-MUT plasmids were mixed with miR-218 mimics and mimics NC were co-transfected into the 293T cells. The miR-218 mimics and mimics NC were purchased from Sangon Biotech Co., Ltd. (Shanghai, China). After transfection of 48 h, cells were harvested and lysed. The luciferase activity was determined by using a dual-luciferase reporter gene assay kit (BioVision, San Francisco, CA, USA). The experiment was conducted in triplicate.
3
Linc00839 Promoter Activity Assay
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The wide type and the mutation of the promoter segment sequences of Linc00839 were synthesized and inserted into a pGL3‐basic vector (Genecreate), respectively. HEK‐293T cells were, respectively, plated in 24‐well plates at 5 × 103 cells per well 8 h before transfection. The cells were co‐transfected with a mixture 2 μg of pGL3‐basic‐Linc00839 promoter, Renilla, and pcDNA3.1‐Myc or control (Dual Luciferase Reporter Gene Assay Kit, Biovision). 48 h later, the luciferase activity was measured using the Dual Luciferase Reporter Assay System (Promega).
4
Runx2 3'UTR Regulatory Mechanism
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The 3′UTR sequences of Runx2 containing miR‐30b binding site were synthesized. Wild‐type (WT) plasmids (Runx2‐WT) and mutant‐type plasmid (Runx2‐MUT) were constructed. Then the constructed vectors were mixed with mimic‐NC or miR‐30b mimic and transfected into 293T cells (American Type Culture Collection, Manassas, Virginia, USA). After 48 h of transfection, luciferase activities were detected using the dual‐luciferase reporter Gene assay kit (BioVision, SanFrancisco, CA, USA) on the Glomax20/20 luminometer (Promega, WI, USA).
5
Transcriptional Regulation of 42Sp50 Promoter
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The possible binding sites of 42Sp50 promoter were predicted by PROMO (http://alggen.lsi.upc.es/cgi-bin/promo_v3/promo/promoinit.cgi?dirDB=TF_8.3 ). The 42Sp50 promoter of spotted scat (3 kb, ligation into Mlu I/Xho I sites) was generated by PCR and subcloned into the pGL3-basic vector (Promega Corp., Madison, WI). Sf1, Foxh1, and Dmrt4 sequence was synthesized by Sangon Biotech and ligated to pcDNA3.1 vector (Invitrogen). The dual-luciferase assay was carried out according to a previous report (Wang et al., 2007 (link)). HEK 293 cells cultured at a density of 2 × 104 cells per well were used for transfection. The reagents used in the experiment include FBS (16140071, Gibco, America), DMEM (SH30022.01, Hyclone, America), streptomycin/penicillin (15070063, Thermofisher, America), Dual-Luciferase Reporter Gene Assay Kit (K801, Biovision, America) and lipo2000 (Invitrogen, America).
6
Verifying HNF4A as a Direct Target of LOC100996425
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The binding sites of LOC100996425 and HNF4A were predicted using a Bioinformatics website Starbase (http://starbase.sysu.edu.cn/index.php ). Dual‐luciferase reporter gene assay was performed to ascertain as to whether HNF4A was a direct target of LOC100996425. The artificially synthesized gene segment of HNF4A 3’‐untranslated region (3’UTR) was introduced into pMIR‐reporter (HUAYUEYANG Technology (Beijing) Co., Ltd.). The mutant (Mut) sequence was designed based on the wild‐type (Wt) HNF4A. Afterwards, the target segments were subsequently inserted into the pMIR‐reporter. The Wt and Mut plasmids with correct sequence were subsequently transfected with LOC100996425 into the human embryonic kidney (HEK)‐293T cells (Shanghai Beinuo Biotechnology). Luciferase activity was detected using a dual‐luciferase reporter gene assay kit (K801‐200, BioVision).
7
Verification of miR-140 binding sites
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The SNHG1 and TLR4 3′-UTR cDNAs comprising the miR-140 recognition site were amplified by PCR, cloned into the pMIR-luciferase reporter vector, and named as SNHG1-wt and TLR4-wt, respectively. In addition, the luciferase reporters SNHG1-mut and TLR4-mut that encompassed the mutated sequence at miR-140 binding site were generated. HCCC-9810 and RBE cells were cotransfected with miR-Ctr or miR-140 and pMIR-luciferase reporter vectors containing the gene fragments of SNHG1 or TLR4 3′UTR with wt or mut binding sites of miR-140 by Lipofectamine 2000. Forty-eight hours after transfection, the luciferase activities were assayed by Dual-Luciferase Reporter Gene Assay kit (BioVision, Milpitas, CA, USA).
8
Luciferase Assay for miR-6881-3p Targeting
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miR‐6881‐3p mimics, miRNA negative control (NC), pmirGLO‐TGFBR1‐wild type (WT)/mutation type (MT), and pmirGLO‐SMAD4‐WT/MT vectors were synthesized by Beijing Syngenbio Co., LTD. HEK‐293T cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM; HyClone) supplemented with 10% fetal bovine serum (FBS, Gibco). Then, HEK-293T cells in the logarithmic growth phase were seeded into 24‐well culture plates and transfected with the corresponding vectors when the cells attained 70%-80% confluence. Various combinations of plasmids are shown in Table 1 . After 48h of transfection, luciferase activities were measured using the Dual‐Luciferase Reporter Gene Assay Kit (Biovision) following the manufacturer’s instructions.
The combinations of transfected plasmids
| Serial number | Plasmid combinations |
|---|---|
| 1 | miR-6881-3p+SMAD4 3’UTR wt |
| 2 | miR-6881-3p+SMAD4 3’UTR mt |
| 3 | miRNA NC+SMAD4 3’UTR wt |
| 4 | miRNA NC+SMAD4 3’UTR mt |
| 5 | miR-6881-3p+TGFBR1 3’UTR wt |
| 6 | miR-6881-3p+TGFBR1 3’UTR mt |
| 7 | miRNA NC+TGFBR1 3’UTR wt |
| 8 | miRNA NC+TGFBR1 3’UTR mt |
9
Identifying miR-3690 Target Genes
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The potential target genes of miR-3690 were predicted using Target Scan Human (version 7.1; http://www.targetscan.org/vert_71 ). Luciferase reporter assays. Based on the binding sites of DDK-3 and miR-3690, which were predicted using TargetScan, wild-type DDK-3 sequences were designed and cloned into the pGL3 luciferase reporter vector (Promega Corporation). Cells were then co-transfected with the vectors and miR-3690, miR-3690-in or miR-3690-mut, using Lipofectamine® 2000 transfection reagent. Following a 48-h incubation, luciferase signals were measured using the Dual luciferase reporter gene assay kit (BioVision, Inc.) according to the manufacturer's protocol. Firefly luciferase activity was normalized to that of Renilla luciferase.
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