Dual glo luciferase assay

Manufactured by Promega

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The Dual-Glo Luciferase Assay is a quantitative luminescent reporter gene assay system. It is designed to measure the activities of firefly and Renilla luciferases simultaneously within a single sample.

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Dual Luciferase Assay (854 citations) Luciferase assay (182 citations) Dual-Glo (44 citations) Dual-Glo Luciferase (24 citations) Dual-Glo luciferase assays system (4 citations)

249 protocols using dual glo luciferase assay

Dual-Glo Luciferase Assay for Herpes Virus

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Dual-Glo luciferase assays (Promega) were employed in co-transfection assays according to the manufacturers’ protocols. To assay for ICP0 function, the LATpICP0 construct (flanked by sequences homologous to the gJ/gD region) that was employed to make the mutants described above was co-transfected with the pRL-TK renilla expression plasmid (promega) or no promoter vector. To assay for VP16 function the LATpVP16 construct employed to make the mutants described above was co-transfected with an ICP0 promoter (124,818 to 124109 BP) firefly luciferase construct or a CMV promoter luciferase construct. Transfection efficiency was determined by including the relevant renilla or firefly luciferase expression plasmids.

Sawtell N.M, & Thompson R.L. (2016). De Novo Herpes Simplex Virus VP16 Expression Gates a Dynamic Programmatic Transition and Sets the Latent/Lytic Balance during Acute Infection in Trigeminal Ganglia. PLoS Pathogens, 12(9), e1005877.

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NFAT-Luciferase Reporter Assay

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HEK-293T cells were seeded in clear-bottom white 96-well plates 20–24 hours prior to transfection. Each well was transfected with 50ng of empty vector (EV) or receptor DNA, 50ng NFAT-luciferase (pGL4.30, Promega, Madison, WI), 1ng Renilla luciferase (pRL-SV40, Promega). Dual-Glo luciferase assays (Promega) were performed 48hr post-transfection and plates were read on a BMG Omega plate reader. The ratio of firefly:Renilla was calculated for each well and normalized to the mean of the EV-transfected controls.

Purcell R.H., Toro C., Gahl W.A, & Hall R.A. (2017). A disease-associated mutation in the adhesion GPCR BAI2 (ADGRB2) increases receptor signaling activity. Human mutation, 38(12), 1751-1760.

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Regulation of NR4A2 3' UTR by miRNAs

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The NR4A2 3′ UTR was cloned by GeneCopoeia, Inc. (Rockville, MD), directly downstream from a firefly luciferase (Fluc) gene under the control of an SV40 promoter in the pEZX-MT01 vector, which also contains a Renilla luciferase (Rluc) gene under the control of a CMV promoter (as a transfection control). This reporter construct (WT 3UTR) was used to identify miRNAs that regulate Fluc activity through binding to the NR4A2 3′ UTR and degradation or translational inhibition of fused Fluc mRNA. The Rluc activity was used to normalize the Fluc. The 293T cells were cotransfected for 48 h with the 3′ UTR reporter plasmid and 75 cancer-related miRNAs selected from a previously described library32 (link) (see Supplementary Table S1) by using Lipofectamine 2000 (Life Technologies) according to the manufacturer’s protocol. Fugene 6 (Promega, Madison, WI) was used for transient cotransfection of reporter gene plasmids and miRNAs into HCT116 cells according to the manufacturer’s instructions. Dual-Glo luciferase assays (Promega, Madison, WI) were performed to measure and calculate the ratios of firefly and Renilla luciferase activity. Luciferase activity was measured with an EnVision 2102 Multilabel Plate Reader (Perkin Elmer Inc., Waltham, MA).

Beard J.A., Tenga A., Hills J., Hoyer J.D., Cherian M.T., Wang Y.D, & Chen T. (2016). The orphan nuclear receptor NR4A2 is part of a p53–microRNA-34 network. Scientific Reports, 6, 25108.

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Dual-Luciferase Assay for NDP/WNT Signaling

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A detailed experimental procedure is described in the supplement. In brief, 293T cells were transiently transfected with receptor complex components, firefly-, and renilla-luciferase constructs. Cells were stimulated with recombinant NDP (R+D Systems) or by co-transfection of NDP or WNT plasmids. Dual-Glo luciferase assays (Promega) were performed. Data were analyzed by calculating the ratio of firefly/renilla luciferase signals and normalizing the data to the data point shown on the left of each bar graph.

Lai M.B., Zhang C., Shi J., Johnson V., Khandan L., McVey J., Klymkowsky M.W., Chen Z, & Junge H.J. (2017). TSPAN12 is a Norrin Co-Receptor that Amplifies Frizzled4 Ligand Selectivity and Signaling. Cell reports, 19(13), 2809-2822.

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Evaluating Compound Activity on CYP3A4 Promoter

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The methods for these procedures were described previously [20 (link)]. Briefly, the cells were transfected with Flag-hPXR, CMV-Renilla, and CYP3A4-luc plasmids by using FuGENE 6 (Roche Diagnostics). After 24 h, cells were seeded in 384-well plates (5000 cells/well) in phenol red–free medium containing 5% charcoal/dextran-treated FBS and incubated for another 24 h before treatment with compounds to be tested. Compounds were transferred by using pintools and incubated with the cells for 24 h before Dual-Glo Luciferase Assays (Promega, Madison, WI) were performed. Renilla luciferase activity was used to normalize the firefly luciferase activity. CYP3A4 promoter activity (percentage of activation; a.u.) was determined as described previously [34 (link)]. Rifampicin (7 μM) and DMSO were used as positive (100%, or a.u. = 100) and negative (0%, or a.u. = 0) controls, respectively in the dose-responsive evaluation of compound activity. Curve-fitting software (GraphPad Prism 4.0; GraphPad Software, La Jolla, CA) was used to generate the curves and to determine the EC₅₀.

Banerjee M, & Chen T. (2014). Thiazide-like diuretic drug metolazone activates human pregnane X receptor to induce cytochrome 3A4 and multidrug-resistance protein 1. Biochemical pharmacology, 92(2), 389-402.

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Quantifying Promoter Activity and VP16 Function

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Dual-Glo luciferase assays (Promega) were employed in co-transfection assays according to the manufacturers’ protocols. To assay for promoter strengths, the constructs in which firefly luciferase was driven by wt or mutant VP16 promoters as well as a cytomegalovirus immediate early gene promoter (CMVIE) driven luciferase construct (Promega) as a positive control, were co-transfected with the pRL-TK renilla expression plasmid (Promega) or no promoter vector. The HSV-2 regulatory sequences were slightly stronger in this assay (≤2-fold higher). To assay for VP16 function, the VP16 constructs employed to make the mutants described above were co-transfected with an ICP0 promoter (124,818 to 124,109 BP) firefly luciferase construct (Sawtell and Thompson, 2016a (link),b (link)). Transfection efficiency was determined and normalized by including the relevant renilla or firefly luciferase expression plasmids (Figure 3A).

Thompson R.L, & Sawtell N.M. (2019). Targeted Promoter Replacement Reveals That Herpes Simplex Virus Type-1 and 2 Specific VP16 Promoters Direct Distinct Rates of Entry Into the Lytic Program in Sensory Neurons in vivo. Frontiers in Microbiology, 10, 1624.

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Quantifying Receptor-Mediated Transcriptional Activity

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HEK-293T/17 cells were seeded in 96-well plates 20–24 h prior to transfection. Each well was transfected with 50 ng of firefly reporter, 1 ng of Renilla luciferase, and 10 ng of either receptor plasmid or empty vector (EV). Reporter constructs (CRE: pGL4.29, Renilla pRLSV40) were acquired from Promega (Madison, WI). After 24–48 hrs, DualGlo luciferase assays (Promega) were performed according to the manufacturer’s protocol and plates were read on a BMG Omega plate reader. Results were calculated for each assay by determining the luminescence ratio of firefly:Renilla luciferase counts, normalized to EV transfected wells. Error bars for all EV-transfected conditions were represented as the standard errors of the normalized raw value means.

Giddens M.M., Wong J.C., Schroeder J.P., Farrow E.G., Smith B.M., Owino S., Soden S.E., Meyer R.C., Saunders C., LePichon J.B., Weinshenker D., Escayg A, & Hall R.A. (2017). GPR37L1 Modulates Seizure Susceptibility: Evidence from Mouse Studies and Analyses of a Human GPR37L1 Variant. Neurobiology of disease, 106, 181-190.

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Cloning and Luciferase Assays of SMAD3 Enhancers

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The genomic regions franking the identified SMAD3 enhancers were PCR-amplified from the genomic DNA of H1299 cells and cloned into pGL3-promoter (Promega). All plasmids were verified by direct DNA sequencing. For reporter assays, 1 × 10⁴ H1299 cells were co-transfected with 50 fmol pGL3 vectors and 2 fmol pRL-TK, a Renilla luciferase expressing vector, using Lipofectamine^TM 3000 according to the manufacturer’s instructions (Invitrogen). Dual-Glo luciferase assays were performed at 48 h post-transfection according to the manufacturer’s instructions (Promega). The activities of Firefly luciferase were normalized with the activities of Renilla luciferase, and results are presented as fold activity to the pGL3-promoter vector alone.

Huang Y.T., Cheng A.C., Tang H.C., Huang G.C., Cai L., Lin T.H., Wu K.J., Tseng P.H., Wang G.G, & Chen W.Y. (2021). USP7 facilitates SMAD3 autoregulation to repress cancer progression in p53-deficient lung cancer. Cell Death & Disease, 12(10), 880.

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Regulation of β-catenin by miR-320a

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Prediction of miR-320a binding sites was performed using TargetScan software (http://www.targetscan.org). Bioinformatics analysis revealed a potential miR-320a binding site for miR-320a in the 3′-UTR region of β-catenin. HEK 293T cells at 60% confluence were transfected with 200 ng DNA from the β-catenin-wild-type (WT)-UTR plasmid (firefly luciferase reporter vector containing the β-catenin 3′-UTR) or β-catenin-WT-UTR DNA (firefly luciferase reporter vector containing the β-catenin 3′-UTR mutant) and 2 ng pRL-TK vector (Promega Corporation, Madison, WI, USA) in combination with miR-320a mimics (final concentration of 100 nM; GenePharma) or miR-NC (100 nM). Transfection was performed with Lipofectamine 2000 (Invitrogen; Thermo Fisher Scientific Inc.). The firefly and Renilla luciferase activities were measured by consecutively using Dual Glo Luciferase assays (Promega Corporation). Firefly l uciferase activity was normalized to Renilla luciferase for each transfected well.

Lu C., Liao Z., Cai M, & Zhang G. (2016). MicroRNA-320a downregulation mediates human liver cancer cell proliferation through the Wnt/β-catenin signaling pathway. Oncology Letters, 13(2), 573-578.

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Survivin 3'UTR Regulation via Luciferase Assay

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The SCC-15 cells were cultured in DMEM supplemented with 10% FBS at 37°C in an atmosphere of 5% CO₂ to 70–80% confluency. Using Lipofectamine 2000 (Invitrogen; Thermo Fisher Scientific, Inc.), the cells were then transfected with the WT-Sur-UTR DNA plasmid, which consisted of a firefly luciferase reporter vector containing the wild-type survivin 3′-UTR, or the mutant Sur-UTR DNA plasmids (Mut1 and Mut2), which consisted of a firefly luciferase reporter vector containing the mutant survivin 3′-UTR, (Fig. 1) and the control pRL-TK vector, which contained Renilla luciferase (Promega Corporation, Madison, WI, USA). The firefly and Renilla luciferase activities were measured consecutively using Dual Glo Luciferase assays (Promega Corporation) 48 h subsequent to transfection.

Ou D., Wu Y., Liu J., Lao X., Zhang S, & Liao G. (2016). miRNA-335 and miRNA-182 affect the occurrence of tongue squamous cell carcinoma by targeting survivin. Oncology Letters, 12(4), 2531-2537.

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