Renilla luciferase construct

Manufactured by Promega

Sourced in United States

The Renilla luciferase construct is a genetic tool used in molecular biology research. It consists of the Renilla luciferase gene, which encodes a bioluminescent protein derived from the sea pansy (Renilla reniformis). The Renilla luciferase construct can be used to report gene expression or monitor cellular processes in various experimental systems.

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Lab products found in correlation

Dual luciferase reporter assay system, by Promega (7 mentions) Dual luciferase reporter system, by Promega (6 mentions) Reporter lysis buffer, by Promega (3 mentions) Dual luciferase assay, by Promega (3 mentions) Lipofectamine 2000 reagent, by Thermo Fisher Scientific (3 mentions) Lipofectamine 2000, by Thermo Fisher Scientific (2 mentions) Luciferase reporter constructs, by Promega (2 mentions) Dual luciferase assay system, by Promega (2 mentions) 6 well plate, by Merck Group (1 mentions) Lipofectaminetm 2000, by Thermo Fisher Scientific (1 mentions) Siportneofx reagent, by Thermo Fisher Scientific (1 mentions) Fugene 6 reagent, by Roche (1 mentions) Accutase, by Merck Group (1 mentions) Fugene 6, by Roche (1 mentions) Dual luciferase reagent, by Promega (1 mentions) Rnaimax, by Thermo Fisher Scientific (1 mentions) Lipofectamine 2000, by Promega (1 mentions) Infinite m200 pro plate reader, by Tecan (1 mentions) Pmirglo, by Promega (1 mentions) Lna inhibitor, by Qiagen (1 mentions)

21 protocols using renilla luciferase construct

NF-κB Luciferase Reporter Assay

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Co-transfection of 200 ng each of a NF-κB p65 luciferase reporter and a Renilla luciferase construct (Promega) was performed on 1×10⁶osteosarcoma cells in each well of a 6-well plate (Sigma) using Lipofectamine^TM 2000 (Invitrogen) according to the manufacturer's instructions. These cells were washed with PBS twice, transfected with siRNA and then lysed using reporter lysis buffer (Promega). For the luciferase assay, a mixture was made at room temperature of 20 μl cell extract and 100 μl luciferase assay reagent. The firefly luciferase activity of this mixture was then quantified using a Dual-Luciferase Reporter Assay System (Promega).

Zeng W., Liu Q., Chen Z., Wu X., Zhong Y, & Wu J. (2016). Silencing of hERG1 Gene Inhibits Proliferation and Invasion, and Induces Apoptosis in Human Osteosarcoma Cells by Targeting the NF-κB Pathway. Journal of Cancer, 7(6), 746-757.

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miRNA Regulation of VPS4a Expression

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HEK 293T cells were co-transfected with either WT or mutated (harboring rs16958754) VPS4a 3′UTR reporter clones for miRNA target validation (Origene) and Renilla luciferase construct (Promega) with miR-16, -103, -107 or a scrambled control mimic according to the manufacturer’s protocol (Life Technologies). The 3′UTR of VPS4a was cloned downstream of the luciferase gene. An Internal ribosome entry site (IRES) is located upstream of luciferase. As the miR/RISC complex binds to the target, luciferase activity is reduced. If there is no interaction between the miR and the target, luciferase activity remains high. Luciferase activity was assessed using the Dual Luciferase Reporter Assay System (Promega) [23] .

Adhikari N., Guan W., Capaldo B., Mackey A.J., Carlson M., Ramakrishnan S., Walek D., Gupta M., Mitchell A., Eckman P., John R., Ashley E., Barton P.J, & Hall J.L. (2014). Identification of a New Target of miR-16, Vacuolar Protein Sorting 4a. PLoS ONE, 9(7), e101509.

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Dual-Luciferase Reporter Assay for Gene Regulation

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NPC cells were co-transfected with a Firefly luciferase reporter construct and the Renilla luciferase construct (Promega). Both luciferase activities were determined 48 h post-transfection using a Dual-luciferase Reporter Assay System (Promega) according to the manufacturer’s instructions. The Firefly luciferase activity was normalized to the Renilla luciferase activity.

Zheng Z.Q., Huang Z.H., Liang Y.L., Zheng W.H., Xu C., Li Z.X., Liu N., Yang P.Y., Li Y.Q., Ma J., Sun Y., Tang L.L, & Wei D. (2023). VIRMA promotes nasopharyngeal carcinoma, tumorigenesis, and metastasis by upregulation of E2F7 in an m6A-dependent manner. The Journal of Biological Chemistry, 299(5), 104677.

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Luciferase Assay for microRNA Activity

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A dual-luciferase reporter system developed by Promega (E1960, Madison, WI, USA) was used to perform luciferase activity assay. Briefly, U343 cells (ATCC, Manassas, VA, USA) were cultured on 12-well tissue culture plates at a density of 2 × 10⁵ cells per well. Cells were co-transfected with the luciferase reporter constructs (Madison, WI, USA), corresponding miRNA mimics, and Renilla luciferase construct (Promega) for 5 h. After 24 h culture, the transfected cells were lysed by 150 μl of passive lysis buffer; 30 μl of lysates were mixed with 50 μl of LAR II, and then firefly luciferase activity was measured by a luminometer. For the internal control, 50 μl of Stop & Glo reagent (Madison, WI, USA) was added to the sample.

Du W.W., Yang W., Fang L., Xuan J., Li H., Khorshidi A., Gupta S., Li X, & Yang B.B. (2014). miR-17 extends mouse lifespan by inhibiting senescence signaling mediated by MKP7. Cell Death & Disease, 5(7), e1355-.

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Transcriptional Regulation of Cathepsin K

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cRel, C/EBPδ, NFAT, and CREB cDNAs were cloned into pCMV4, and hnRNP A2 cDNA into pCI, as described before.^{22 (link)} Transfections of cDNAs were carried out in cells grown on 6-well dishes using FuGENE® 6 reagent (Roche Molecular Biochemicals, Indianapolis, IN). One µg of promoter DNA construct (pGL3-Cath K) and 0.5 µg of a Renilla luciferase construct (Promega), as an internal control, were used in each transfection. Luciferase activity was assayed using the Dual-Luciferase® reporter assay system (Promega). Cotransfections with various cDNAs were carried out using 0.2 µg of each cDNA construct.
For mRNA silencing by transient transfections, predesigned siRNA for mouse C/EBPδ (sc-37723) (Santa Cruz Biotechnology, Santa Cruz, CA), shRNA specific for hnRNPA2, and double-stranded scrambled negative siRNA control (Integrated DNA Technologies, San Diego, CA) were used as described earlier.^{22 (link)} Cells (1 × 10⁶) were transfected with preannealed double-stranded siRNAs for 48 h at a final concentration of 25 nM by reverse transfection as described before.^{22 (link)} Transient transfections were carried out in triplicate by using siPORT™ NeoFX™ reagent (LifeTechnologies). After 48 hours, cells were lysed and cathepsin K mRNA expression was assessed by real time PCR.

Guha M., Srinivasan S., Koenigstein A., Zaidi M, & Avadhani N.G. (2015). Enhanced osteoclastogenesis by mitochondrial retrograde signaling through transcriptional activation of the cathepsin K gene. Annals of the New York Academy of Sciences, 1364(1), 52-61.

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Transient Transcription Assay for RARα

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COS-7 cells were transfected with RARα1, RARα2 and PML-RAR, alone or in combination in the presence of the RARE-containing β2RARE-luciferase reporter [64 (link)]. The normalization plasmid is a renilla luciferase construct (Promega) [63 (link)].

Gianni M., Fratelli M., Bolis M., Kurosaki M., Zanetti A., Paroni G., Rambaldi A., Borleri G., Rochette-Egly C., Terao M, & Garattini E. (2016). RARα2 and PML-RAR similarities in the control of basal and retinoic acid induced myeloid maturation of acute myeloid leukemia cells. Oncotarget, 8(23), 37041-37060.

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Assessing β-Catenin Transcription Activity

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β-Catenin transcription was assessed using the TOP FLASH/FOP FLASH reporter luciferase assay.^{24 (link)} For TOP FLASH/FOP FLASH reporter luciferase assay, hNPCs were dissociated with Accutase (37 °C 15 min; Sigma) and plated onto poly-D-lysine-coated 24-well plates at 10⁵ cells per well in differentiation medium. WNT pathway was stimulated adding 1.5 μM CHIR (WNT1 agonist, TOCRIS Biosciences, Minneapolis, MN, USA) at the time of plate down and kept in the medium until the cells were harvested. The following day cells were co-transfected using Fugene 6 (Roche) with 0.99 μg of β-catenin-LEF/TCF-sensitive (TOP) or β-catenin-LEF/TCF insensitive (FOP) reporter vector (Addgene) together with 0.1 μg of a constitutively active Renilla luciferase construct (Promega) for normalization of transfection efficiency. Cells were processed 48 h after co-transfection for Luciferase reporter activity using the Dual Luciferase Reporter System (Promega) according to the manufacturer's instructions.

Rinaldi F., Hartfield E.M., Crompton L.A., Badger J.L., Glover C.P., Kelly C.M., Rosser A.E., Uney J.B, & Caldwell M.A. (2014). Cross-regulation of Connexin43 and β-catenin influences differentiation of human neural progenitor cells. Cell Death & Disease, 5(1), e1017-.

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NUCKS-Mediated Luciferase Reporter Assay

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NUCKS (1 ug; Promega) pISRE-Luc (0.3 μg; Strategene) and Renilla luciferase construct (0.2 μg; Promega) were transfected with Lipofectamine 2000 (Invitrogen) and siRNA(50 nM; Dharmacon and Qiagen) with RNAiMax (Invitrogen). Cells were stimulated with the indicated ligands 24 hours post-transfection and assayed for luciferase activity at particular time points following stimulation by Dual-luciferase reagent (Promega) according to the manufacturer’s instructions.

Poon M.W., Jiang D., Qin P., Zhang Y., Qiu B., Chanda S., Tergaonkar V., Li Q., Wong I.Y., Yu Z., Tse H.F., Wong D.S, & Lian Q. (2017). Inhibition of NUCKS Facilitates Corneal Recovery Following Alkali Burn. Scientific Reports, 7, 41224.

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Luciferase Assay in Macrophage Subtypes

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We cotransfected macrophages (M0, M1-like TAMs, and M2-like TAMs) in six-well plates using TCF/LEF luciferase construct (0.3 μg per well) and Renilla luciferase construct (10 ng per well; Promega) using Lipofectamine 2000 transfection reagent for 6 hours in optimum serum-free medium. After 6 hours, we incubated cells with serum-containing medium for 24 hours at 37°C. We quantified luciferase activities using the dual-luciferase reporter assay system (Promega) according to the manufacturer’s instructions and a spectrofluorometer (Tecan Infinite M200 PRO plate reader). The ratio of luciferase signal–to–Renilla signal for each well was calculated as previously described (42 (link)).

Sarode P., Zheng X., Giotopoulou G.A., Weigert A., Kuenne C., Günther S., Friedrich A., Gattenlöhner S., Stiewe T., Brüne B., Grimminger F., Stathopoulos G.T., Pullamsetti S.S., Seeger W, & Savai R. (2020). Reprogramming of tumor-associated macrophages by targeting β-catenin/FOSL2/ARID5A signaling: A potential treatment of lung cancer. Science Advances, 6(23), eaaz6105.

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Transfection and Luciferase Assay for Cd274 Regulation

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Transient transfection was performed using Lipofectamine 2000 (Invitrogen). For promoter study, B16, pre- or post-differentiated 3T3-L1 cells grown in 24-well plates were co-transfected with 0.1 μg of pGL3 basic empty control plasmid (Promega, Cat: #E1751) or pGL3-Cd274 promoter construct and 0.02 μg of the Renilla luciferase construct (Promega, Cat: #E6911, for normalization of transfection efficiency). For 3’UTR study, pmirGLO (Promega, Cat: #E1330) or pmirGLO-Cd274 3’UTR vector were transfected into pre- or post-differentiated 10T1/2 cells. Luciferase activity was detected by Promega dual-luciferase reporter assay system (Promega, Cat: # E1910).

Wu B., Sun X., Gupta H.B., Yuan B., Li J., Ge F., Chiang H.C., Zhang X., Zhang C., Zhang D., Yang J., Hu Y., Curiel T.J, & Li R. (2018). Adipose PD-L1 Modulates PD-1/PD-L1 Checkpoint Blockade Immunotherapy Efficacy in Breast Cancer. Oncoimmunology, 7(11), e1500107.

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