All experimental cells were, separately, seeded into 12-well plates (1.5 × 105 cells/well), and allowed for growth to reach 80% of confluence, before the cells were co-transfected using a lipofectamine 3000 mixture with each of ARE (antioxidant response elements)-driven luciferase plasmids (which were made by inserting each of the indicated ARE sequences into the pGL3-Promoter vector) or non-ARE reporter plasmids (as an internal background control), together with an experiment construct for Nrf1, Nrf2, or an empty pcDNA3.1 vector. In this test, the Renilla expression by pRL-TK (a plasmid encoding renilla luciferase driven by the thymidine kinase promoter) served as an internal quality control for transfection efficiency. Thereafter, the luciferase activity was measured by the dual-luciferase reporter system (Beyotime, Shanghai, China). The resulting data were calculated as fold changes (mean ± SD, n = 3 × 3) relative to the controls, which are representative of at least three independent experiments being each performed in triplicates.
Dual luciferase reporter system
Manufactured by Beyotime
Sourced in China
The Dual-luciferase reporter system is a tool used to measure and compare the activity of two different luciferase reporter enzymes simultaneously. It provides a quantitative analysis of gene expression and regulation.
Automatically generated - may contain errors
Lab products found in correlation
Bhp9504 optical analysis system, by Hamamatsu Photonics (2 mentions)
Pgl3 basic plasmid, by Promega (2 mentions)
Quickchange site directed mutagenesis kit, by Agilent Technologies (2 mentions)
Lipofectamine 2000, by Thermo Fisher Scientific (2 mentions)
Dual luciferase reporter vector, by Hanbio Biotechnology (1 mentions)
Pgl3 luciferase vector, by Promega (1 mentions)
Pgl4 vector, by Promega (1 mentions)
Pgl3 luciferase promoter vector, by Promega (1 mentions)
P mir reporter plasmid, by Thermo Fisher Scientific (1 mentions)
Lipofectamine 3000, by Beyotime (1 mentions)
20 protocols using dual luciferase reporter system
1
Dual-Luciferase Assay for Nrf Transcriptional Activity
2
Luciferase Assay for miR-139-5p
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HCT-116 cells were co-transfected with pLuc, pRL-CMV, miR-139-5p mimic (negative control, NC), and pcDNA3.1-RP11-757G1.5 (pcDNA3.1-) and then subjected to luciferase assays using the dual-luciferase® reporter system (Beyotime, Cat. No. E1960).
3
Validating HOXC9 as a miR-26a Target
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The miR-26a mimics (5'-TTCAAGTAATCCAGGATAGGCT-3'), dual-luciferase reporter vector, and Lipofectamine 2000 reagent were purchased from Hanbio technology. The 3ʹ-UTR of HOXC9 containing the putative binding sites (5'-TACTTGA-3') was cloned downstream of the luciferase gene to construct a wild-type plasmid (WT). The 3ʹ-UTR containing mutated binding sites (5'-ATGAACT-3') was cloned into the luciferase gene to construct a mutant plasmid (MUT). HEK293 cells were cultured and co-transfected with miR-26a mimics, the WT plasmid, or the MUT plasmid. After 24 h, both firefly and Renilla luciferase activity were detected using a dual-luciferase reporter system (BeyotimeBio, Shanghai, China). The firefly luciferase activity was normalized to the Renilla luciferase activity [17] .
4
Analyzing miR132-3p Target Gene
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HepG2 cells (2×105/well) were seeded in 12-well plates. The next day cells were co-transfected with 1 µg pYr-MirTarget-Sox4-3′UTR reporter plasmid, 50 nmol/l of miR132-3p mimic or mimic control using Lipofectamine 2000. Then, 24 h after transfection, both Firefly and Renilla luciferase activities were quantified using the dual luciferase reporter system (Beyotime Institute of Biotechnology) according to the manufacturer's instructions. The relative luciferase unit (Renilla luciferase/Firefly luciferase) was calculated to determine the activation of the target gene. All experiments were performed at least twice.
5
Regulation of Choline Acetyltransferase by HIF-1α
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Chat activity was detected by a dual-luciferase reporter system (Beyotime, Shanghai, China)54 (link). Cultured NSCs from Ddah1 WT, KO and TG mice were transfected with Lipofectamine 2000 reagent with the reporter construct pGL4.27 containing the Chat gene promoter. The pRL-CMV plasmid, which expressed Renilla luciferase, was used as an internal control to normalize the transfection efficiency. All transfections were normalized to the total equal amount of DNA with the empty plasmid pGL4.27. To observe the regulation of Chat by HIF-1α, KC7F2 (20 μmol/L), an inhibitor of HIF-1α, was applied to NSCs isolated from Ddah1 TG mice during and after transfection. Twenty-four hours after transfection, the cells were harvested, and the luciferase activity of Chat was measured using a SpectrMax M2e luminometer (Molecular Devices, San Jose, CA, USA).
6
Luciferase Assays for Promoter Activity
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Luciferase assays were performed as described previously (52 (link)). Briefly, HEK293T cells were transfected with pGL3 vectors containing the promoter of il10 or il6, vectors encoding RelB or p65, and a control reporter plasmid encoding EF1 promoter-driven Renilla luciferase. The transfected cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM) containing 10% FBS for 24 hours and then collected and lysed. Firefly and Renilla luciferase activities were measured with the Dual-Luciferase Reporter System (Beyotime Biotechnology), and Renilla luciferase activity was used to normalize for transfection efficiency.
7
BRE-Luc Reporter Lentivirus Transduction
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hMSCs were infected with BRE-Luc reporter lentivirus containing BREs from the Id1 promoter fused to a luciferase reporter gene (Korchynskyi and ten Dijke, 2002 (link); Yadav et al., 2012 (link)). 2 d later, the reporter cells were infected with various lentiviral vectors as specified in each experiment. To measure luciferase activities, the cells were harvested, suspended in culture medium, and added to wells (100 µl containing 104 cells/well) of 96-well white wall plates. The plates were incubated at 37°C in a humidified cell culture incubator with 5% CO2 for 2–3 d. Luciferase activities were analyzed with the Dual-Luciferase Reporter System (Beyotime Biotechnology). The Renilla luciferase activities were used as internal controls, and values of firefly luciferase activities were normalized to the corresponding Renilla luciferase activities. All experiments were repeated at least three times.
8
Validating miR-125b Regulation of VDR
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TargetScan was used to predict miRNAs that could potentially target VDR and identify possible binding regions. The VDR 3'-untranslated region (UTR) contained miR-125b binding sites. The dual-luciferase reporter system (Beyotime, Shanghai, China) was then used to verify luciferase activity. The VDR 3'-UTR cDNA sequence, including the mutant or wild-type miR-125b binding region, was amplified and cloned into the pGL3 luciferase vector (Promega, Madison, WI, USA). Next, 789-O cells were transfected with luciferase reporter plasmids and ether miR-125b inhibitor or NC using Lipofectamine 2000, according to the manuscript protocol. Then, the activity of luciferase was determined using a luminometer (Promega, Madison, WI, USA) and measured based on that of the empty pGL3 vector.
9
Assaying Spry1 Promoter Activity
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The luciferase reporter plasmids pGL3-Basic, pGL3-Spry1, and pCDH-Dmrt1 were stored at the Shaanxi Centre of Stem Cells Engineering & Technology, Northwest A&F University. The vectors were constructed by cloning the promoter of Spry1 into the pGL3-Basic plasmid (Promega, USA). A total of 50 ng of pGL3-Basic vector or pGL3-Spry1-promoter supplemented with pCDH-Dmrt1 was co-transfected into TM4 cells in a 48-well plate using transfection reagent Lipo6000, followed by incubation in Opti-MEM for 30 min at 37 °C. A dual-luciferase reporter system (Beyotime, China) was used to evaluate promoter activity (as determined by fluorescence levels) according to the manufacturer’s instructions. Fluorescence levels were assessed using the BHP9504 optical analysis system (Hamamatsu Photonics, Japan) (Wei et al., 2021 (link)).
10
CircHIPK3 Luciferase Reporter Assay
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The potential target miRNAs of circHIPK3 were predicted using the online database starBase v3.0 (http://starbase.sysu.edu.cn/ ). For the dual-luciferase reporter assay, the segmental sequence of circHIPK3 embracing the predicted binding sites of miR-381-3p was inserted into the pGL4 vector (Promega, Madison, WI, USA) to build wild-type luciferase reporter of circHIPK3 (wt-circHIPK3). Quick Change Site-Directed Mutagenesis kit (Agilent Technologies, Inc., Santa Clara, CA, USA) was used to mutate the binding sites, then mutant-type luciferase reporter of circHIPK3 (mut-circHIPK3) was compounded in a similar way. H1975 and A549 cells were introduced with wt-circHIPK3 or mut-circHIPK3 and miR-381-3p or miR-NC. After 48 h, relative luciferase activity was determined using the Dual-Luciferase reporter system (Beyotime) referring to the user’s manual.
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