The c-Myc regulatory element sequence was synthesized by GENEWIZ and cloned into the pGL4.20 vector. pcDNA3.1-c-Myc, pGL4.20-c-Myc-luc, and Renilla were transfected into HEK293T cells and subsequently treated with a given dose of compound D347-2761 for 24 h. Then the samples were prepared and carried out by Dual-Lumi™ II Luciferase Reporter Gene Assay Kit (Beyotime Biotechnology) according to the manufacturer’s protocol.
Dual lumi 2 luciferase reporter gene assay kit
Manufactured by Beyotime
Sourced in China
The Dual-Lumi™ II Luciferase Reporter Gene Assay Kit is a tool designed for the quantitative analysis of reporter gene expression. It provides a sensitive and efficient method for measuring the activity of firefly and Renilla luciferase reporter genes, which are commonly used as indicators of gene expression and cellular activity.
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Lab products found in correlation
Lipofectamine 3000 reagent, by Thermo Fisher Scientific (3 mentions)
Lipofectamine 3000, by Thermo Fisher Scientific (2 mentions)
Fluorescent microplate reader, by Thermo Fisher Scientific (1 mentions)
Psi check2 plasmid, by Hanbio Biotechnology (1 mentions)
Lipofiter 3, by Hanbio Biotechnology (1 mentions)
Glomax microplate luminometer, by Promega (1 mentions)
V790 20, by Thermo Fisher Scientific (1 mentions)
Pmirglo dual luciferase mirna target expression vector, by Promega (1 mentions)
Lipo8000 transfection reagent, by Beyotime (1 mentions)
Pmir report luciferase plasmid, by Thermo Fisher Scientific (1 mentions)
Dual luciferase detection kit, by Beyotime (1 mentions)
Lipofectamine 2000, by Thermo Fisher Scientific (1 mentions)
293t cells, by Hanbio Biotechnology (1 mentions)
Pgl4.10 vector, by Promega (1 mentions)
Glomax 20 20 luminometer, by Promega (1 mentions)
Cell lysis buffer, by Beyotime (1 mentions)
β catenin sirna, by Genomed (1 mentions)
Prl tk renilla plasmid, by Promega (1 mentions)
Mithras lb940 multilabel reader, by Berthold Technologies (1 mentions)
Dual luciferase reporter assay system, by Promega (1 mentions)
15 protocols using dual lumi 2 luciferase reporter gene assay kit
1
Investigating c-Myc Transcriptional Regulation
2
Dual Luciferase Reporter Assay Protocol
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Dual luciferase reporter assay was performed using Dual-Lumi™ II Luciferase Reporter Gene Assay Kit (Beyotime Biotechnology) and a fluorescent microplate reader (Thermo Fisher Scientific). All plasmids were constructed by SyngenTech (Beijing, China) and transfected into cells. Their sequences were listed in
Supplementary Table S2 . After 24 h, the cells were lysed, centrifuged (10,000‒15,000
g, 3‒5 min), and the supernatant was retained. A total of 100 μL firefly luciferase detection reagent was added to every 20 μL sample, incubated at room temperature for 5 min and then detected using a fluorescent microplate reader, with the result recorded as fLuc. Subsequently, 100 μL renilla luciferase detection reagent was added to the sample and mixed well. The results were recorded as rLuc using a fluorescent microplate reader. The results were presented as the relative expression levels of fLuc and rLuc (fLuc /rLuc ratios).
g, 3‒5 min), and the supernatant was retained. A total of 100 μL firefly luciferase detection reagent was added to every 20 μL sample, incubated at room temperature for 5 min and then detected using a fluorescent microplate reader, with the result recorded as fLuc. Subsequently, 100 μL renilla luciferase detection reagent was added to the sample and mixed well. The results were recorded as rLuc using a fluorescent microplate reader. The results were presented as the relative expression levels of fLuc and rLuc (fLuc /rLuc ratios).
3
Validating miR-370-3p Binding Sites
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In the psiCHECK2 plasmid (HanBio, Shanghai, China), the target circIgfbp2 sequence or the target BACH1 mRNA 3’UTR sequence was cloned downstream of the hRluc gene to construct two reporter vectors containing the potential binding site of miR-370-3p. Next, two different reporter vectors and miR-370-3p mimics were co-transfected into 293 T cells, and Renilla luciferase activity and firefly luciferase activity were determined by Dual-Lumi™ II Luciferase Reporter Gene Assay Kit (BeyoTime, China). Relative luciferase activity = Renilla luciferase activity/firefly luciferase activity.
4
Evaluating FOXA2 Overexpression on IL-6 Luciferase Activity
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The 293T and 16HBE cells were seeded into 96-well plates. They were transfected with IL-6 luciferase reporters, the pRL-TK vector, and the FOXA2 overexpression plasmid or blank pcDNA using LipoFiter3.0 (Hanbio). Twenty-four hours later, the medium was removed, and the cells were collected for luciferase activity measurements using the Dual-Lumi™ II Luciferase Reporter Gene Assay Kit (Beyotime) according to the manufacturer’s instructions. Firefly luciferase values were normalised to the Renilla luciferase values.
5
Luciferase Reporter Gene Assay
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The Dual-Lumi II Luciferase Reporter Gene Assay Kit (Beyotime, Shanghai, China) was used in this experiment. Y5Y-APP cells were seeded in a 96-well plate for 24 h before transfection. After 48 h of transfection, luciferase activity was measured by a GloMax microplate luminometer (Promega, Madison, WI, USA) according to the instructions of the manufacturer [35 (link)].
6
Luciferase Assay of Lsd-1, regucalcin, yip2, CG5162
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Full-length cDNAs of Lsd-1, regucalcin, yip2, and CG5162 were cloned into the pmirGLO dual-luciferase miRNA target expression vector (E1330, Promega, Beijing, China). The full-length cDNA of arlr was cloned into a pcDNA3.1 vector (V790-20, Invitrogen). HEK293T cells (CRL-3216, ATCC, USA) were cultured in DMEM medium (11965-092, Gibco, New York, USA) with 10% fetal bovine serum (11011-8611, Biobase, Jinan, China) and 1% antibiotic (15140-122, Gibco), and maintained in incubators with 5% CO2 at 37 °C. Cells were seeded in a 24-well plate. Transfection was performed at 70–90% confluence with Lipo8000 transfection reagent (C0533, Beyotime). Luciferase assays were performed 2 days after transfection using Dual-Lumi II luciferase reporter gene assay kit (RG089S, Beyotime). Results were tested by SpectraMax i3x (TECAN, Switzerland).
7
PPARγ Luciferase Assay in MH-S Cells
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MH-S Cells were transfected with PPARγ luciferase reporter plasmid (YEASEN) for 48 h. The luciferase activities were measured using Dual-Lumi™ II Luciferase Reporter Gene Assay Kit (Beyotime).
8
BAG5 3'UTR Luciferase Assay
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BAG5 was predicted to be a target gene of miR-142-5p based on analysis with TargetScan (http://www.targetscan.org , v7.2), which yielded a context++ score (The context++ score for a specific site is the sum of the contribution of a series of features). The 3'-untranslated region (3'UTR) of BAG5 was amplified via PCR from bovine mammary epithelial MAC-T cell cDNA and then inserted into the multiple cloning site downstream of the luciferase reporter gene in the pMIR-REPORT™ luciferase plasmid (Thermo Fisher Scientific, Inc.) to construct the luciferase reporter plasmid [BAG5 3'UTR wild-type (WT)]. MAC-T cells were transfected with 1 µg 3'UTR WT or mutant (MUT) constructed by chemical synthesis (General Biosystems Co., Ltd.) and 50 nM miR-142-5p mimics or miR-142-5p mimics NC using Lipofectamine® 2000 (Invitrogen; Thermo Fisher Scientific, Inc.) according to the manufacturer's protocol. Cells transfected BAG5 3'UTR WT or MUT plasmid served as blank group. After 24 h incubation at 37˚C, the cells were lysed by lysis buffer as supplied by the Dual-Luciferase Detection kit (Beyotime Institute of Biotechnology) on ice and luciferase activity was measured using a Dual-Lumi II Luciferase Reporter Gene Assay kit (Beyotime Institute of Biotechnology) according to the manufacturer's instructions. Firefly luciferase activities were normalized to Renilla luciferase activities.
9
Validation of miRNA-Target Interactions
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The target circLphn3 sequences or target mRNA (ZO-1) 3′ untranslated region (UTR) sequences were cloned downstream of hRluc in psiCHECK2 plasmid to construct vectors containing potential miR-185-5p-binding sites. The reporter vectors (HanBio) and miR-185-5p mimics (HanBio) were co-transfected into 293T cells (HanBio) and the renilla luciferase activity (reporter gene) and firefly luciferase activity (internal reference gene) were determined by the Dual-Lumi™ II Luciferase Reporter Gene Assay Kit (BeyoTime). Relative luciferase activity = renilla luciferase activity/firefly luciferase activity.
10
Characterizing the OSR2 Promoter Activity
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All plasmids were obtained from MLPSP (China). OSR2 promoter regions, ranging from 2000 bp upstream of the transcriptional start site to 100 bp downstream, amplified from human genomic DNA using PCR, were inserted into upstream of luciferase gene of pGL4-Basic vector, respectively. The sequences of the plasmids were confirmed by plasmid DNA sequencing. For eliminating differences in transfection efficiency or cell number, pGL4.74 (hRluc-TK) encoding Renilla luciferase was employed as an internal reference. HEK293T cells were cultured in DMEM (Hyclone, USA) containing 10% FBS (Hyclone, USA) followed by used for luciferase reporter assay. HEK293T cells were subjected into 24-well plates. Each plate was transiently co-transfected by these two vectors with or without a MAX overexpression vector. Transfection of all plasmids into HEK293T cells was performed by utilizing Lipofectamine™ 3000 reagent (Invitrogen, USA). Assays were performed three biological replicates by utilizing Dual-Lumi™ II Luciferase Reporter Gene Assay Kit (Beyotime Biotechnology, China).
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