Prl tk renilla luciferase construct

Manufactured by Promega

Sourced in United States

The PRL-TK Renilla luciferase construct is a reporter gene system that utilizes the Renilla luciferase enzyme as a reporter. The Renilla luciferase gene is under the control of the Thymidine Kinase (TK) promoter. This construct can be used to monitor gene expression and regulatory activities in various experimental systems.

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PRL-TK Renilla construct PRL-Renilla luciferase construct PRL-TK Renilla luciferase PRL-TK construct PRL-TK Renilla Renilla luciferase construct Renilla-luciferase (pRL) PRL-TK Renilla construct Renilla-TK

17 protocols using prl tk renilla luciferase construct

Cotransfection and Luciferase Assays for PR-B and FOXO1 Analysis

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Cells were cotransfected overnight using FuGene HD^® transfection reagent (Roche) according to manufacturer’s instructions with either 0.5 µg of a PRE-containing^{31 (link)} (2X-PRE) firefly luciferase reporter construct, 1 µg of a p21 promoter-containing firefly luciferase reporter construct^{21 (link)}, or 4 µg (for p21 promoter-luciferase assay) or 0.5 µg (for RT-qPCR analysis of endogenous genes in PR-B cells) of GFP-tagged N3-PRA vector. The constitutively active pRL-TK-Renilla luciferase construct (Promega, #E2241) (10 ng) was cotransfected as a transfection control. Luciferase assays were performed as previously described^{21 (link)} using the dual luciferase reporter assay (Promega, #E1910).
In experiments using HeLa cells, 10 ng of GFP-tagged EV or PR-A vector, 10 ng pcDNA3 empty vector (pc-EV) or pcDNA3 Flag FKHR^{46 (link)} (pc-FOXO1 WT), 0.5 µg of a PRE-containing^{31 (link)} (2X-PRE) firefly luciferase reporter construct, and 10 ng of a constitutively active pRL-TK-Renilla luciferase construct (Promega, #E2241) were transiently cotransfected. pcDNA3 Flag FKHR was a gift from Kunliang Guan (Addgene plasmid # 13507).
One microgram of pcDNA3 empty vector, pcDNA3 Flag FKHR WT, or Flag FKHR AAA^{46 (link)} were transiently transfected in PR-B+ (clone #1) cells. pcDNA3 Flag FKHR WT and FKHR AAA was a gift from Kunliang Guan (Addgene plasmid # 13507, 13508).

Diep C.H., Knutson T.P, & Lange C.A. (2015). Active FOXO1 is a Key Determinant of Isoform-Specific Progesterone Receptor Transactivation and Senescence Programming. Molecular cancer research : MCR, 14(2), 141-162.

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Allelic Variant Luciferase Assay

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Both allelic forms (QQ and qq) of the 1010 bp open chromatin fragment were synthesized and cloned into the pGL3-basic and pGL3-promoter luciferase reporter vectors (Promega Corporation). The sequence and orientation of the inserts were confirmed by sequencing. For cell culture, DF1 (a chicken fibroblast cell line) cells were cultured in 24-well plates with DMEM (Gibico, Carlsbad, CA, USA) supplemented with 10% FBS (Gibico, Carlsbad, CA, USA) in a 37 °C incubator with 5% CO₂. Using Lipofectamine 2000 (Invitrogen, Carlsbad, CA) according to the manufacturer’s recommendations, we transfected per well cell in a 24 well plate (~80%–90% confluency) with a mixture comprising 720 ng of the pGL3 firefly luciferase reporter construct, 90 ng of the pRL-TK Renilla luciferase construct (Promega Corporation) and 3 µl Lipofectamine 2000. The luciferase assay was performed 48 h after transfection using the Dual Luciferase Reporter Assay system (Promega Corporation) and an Infinite F200 Luminometer (Tecan, Switzerland). Ratios of firefly luminescence/Renilla luminescence were calculated. For each test construct, one expression value was obtained as the average of three technical replicates in each plate (Supplementary Data 11).

Wang Y., Cao X., Luo C., Sheng Z., Zhang C., Bian C., Feng C., Li J., Gao F., Zhao Y., Jiang Z., Qu H., Shu D., Carlborg Ö., Hu X, & Li N. (2020). Multiple ancestral haplotypes harboring regulatory mutations cumulatively contribute to a QTL affecting chicken growth traits. Communications Biology, 3, 472.

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RelB Promoter Luciferase Assay

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The RelB promoter containing 898 base pairs of sequence directly upstream of the start site was cloned into the PGL3-Basic vector (Promega) using SacI and XhoI restriction enzymes (New England Biolabs). 293 cells were transfected with 500 ng of the RelB reporter construct, 50 ng pRL-TK Renilla luciferase construct (Promega) and 500 ng of either pFLAG-CMV-p65 or pCMVHA-hEZH2 (Addgene, #24230) using polyethylenimine linear (Sigma-Aldrich). The cells were harvested 24 hours later in passive lysis buffer and analyzed according to Dual-Luciferase Assay System protocol (Promega). Activity was measured using an Lmax Luminometer (Molecular Devices) and relative light units were normalized to pRL-TK Renilla luciferase light units.

Lawrence C.L, & Baldwin A.S. (2016). Non-Canonical EZH2 Transcriptionally Activates RelB in Triple Negative Breast Cancer. PLoS ONE, 11(10), e0165005.

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Transcriptional Regulation of MTP Genes

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The following 5′-untranslated regions (UTRs) were subcloned downstream of an SV40 promoter and upstream of the firefly luciferase gene in a pGL3-Control vector (Promega): 1) 5′-UTR for MTP-A; 2) 5′-UTR for MTP-A plus first four bases (ATGA) of coding sequence of MTP; 3) 5′-UTR for MTP-C; 4) 5′-UTR for MTP-C plus first four bases (ATGA of coding sequence of MTP. HEK 293 cells were transfected with pGL3-Control or one of the 5′-UTR pGL3 reporter constructs (above), along with pRL-TK renilla luciferase construct (Promega) using FuGENE 6. The cells were harvested three days after transfection and assayed for luciferase activity using a Dual-Glo Luciferase Assay System (Promega).

Suzuki T, & Swift L.L. (2016). Discovery of Novel Splice Variants and Regulatory Mechanisms for Microsomal Triglyceride Transfer Protein in Human Tissues. Scientific Reports, 6, 27308.

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ALKBH5 and P53 Transcriptional Regulation

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PC cells were transfected with a luciferase reporter, the pRL-TK Renilla luciferase construct (Promega), and the ALKBH5 or P53 expression vectors. The ratios of firefly and Renilla luciferase activities were determined 48 h post-transfection using a Dual-Luciferase Assay kit (Promega).

Guo X., Li K., Jiang W., Hu Y., Xiao W., Huang Y., Feng Y., Pan Q, & Wan R. (2020). RNA demethylase ALKBH5 prevents pancreatic cancer progression by posttranscriptional activation of PER1 in an m6A-YTHDF2-dependent manner. Molecular Cancer, 19, 91.

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Evaluating p53 Reporter Activity

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Siha-mock-shRNA/OVA12-shRNA, MCF-7-mock-shRNA/OVA12-shRNA, and Caski-mock/OVA12 cells were plated in 6-well plates and co-transfected with 4.0 μg of the p53 reporter plasmid pp53-TA luc and 0.4 μg of pRL-TK Renilla luciferase construct (Promega) as an internal control using Lipofectamine 2000. After a 48-h incubation, cell lysates were harvested in 500 μl of Passive Lysis Buffer, and the p53 activity was determined with a luciferase assay system (Promega) according to the manufacturer's instructions. All experiments were performed in triplicate.

Zhang R., Wu X., Xia X., Khanniche A., Song F., Zhang B., Wang Y, & Ge H. (2017). OVA12 promotes tumor growth by regulating p53 expression in human cancer cells. Oncotarget, 8(32), 52854-52865.

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Dual-luciferase Assay for TCF/LEF Promoter Activity

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Dual-luciferase assays were performed using the Dual-Luciferase Reporter Assay System (Promega) according to the manufacturer’s instructions. The TCF/LEF reporter plasmid construct (Promega) was transfected into 1 × 10⁶ cells by electroporation, and the measurement performed after 24 hours. The pRL-TK renilla luciferase construct (Promega) was used as an internal control for transfection efficiency. Both firefly and renilla luciferase activity were measured using a GloMax® 20/20 Luminometer (Promega). The ratio of firefly to renilla luciferase activities was used as the TCF/LEF promoter activity.

Takahashi Y., Hiratsuka S., Machida N., Takahashi D., Matsushita J., Hozak P., Misteli T., Miyamoto K, & Harata M. (2020). Impairment of nuclear F-actin formation and its relevance to cellular phenotypes in Hutchinson-Gilford progeria syndrome. Nucleus, 11(1), 250-263.

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Androgen Receptor Transcriptional Activity Assay

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The androgen-responsive element (ARE)-luciferase reporter plasmid, containing three repeats of the ARE region ligated in tandem to the firefly luciferase reporter [38] (link), was used to measure AR trans-activating activity. It was transiently co-transfected into cells with the pRL-TK Renilla luciferase construct (Promega) at 20∶1 ratio as described [39] (link). Dual luciferase assay was conducted per manufacture's instruction (Promega), and the activity of the firefly luciferase was normalized to that of the Renilla luciferase.

Cao B., Qi Y., Yang Y., Liu X., Xu D., Guo W., Zhan Y., Xiong Z., Zhang A., Wang A.R., Fu X., Zhang H., Zhao L., Gu J, & Dong Y. (2014). 20(S)-Protopanaxadiol Inhibition of Progression and Growth of Castration-Resistant Prostate Cancer. PLoS ONE, 9(11), e111201.

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Dual Luciferase Assay for FOXP1

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HEK293 cells were transfected with 50 ng of pGL3-promoter construct (Promega), 50 ng of pRL-TK renilla luciferase construct (Promega) and either empty pNTAP-B vector, TAP-tagged FOXP1-WT or TAP-tagged FOXP1-K670R. Cells were lysed 48 hr after transfection and both firefly and renilla luciferase activity quantified using the Dual Luciferase Reporter Assay System (Promega).

Rocca D.L., Wilkinson K.A, & Henley J.M. (2017). SUMOylation of FOXP1 regulates transcriptional repression via CtBP1 to drive dendritic morphogenesis. Scientific Reports, 7, 877.

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Investigating Transcriptional Regulation in NRVMs

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NRVMs were serum starved overnight (1% FBS/DMEM) followed by transient transfection with 200ng of pGL3 control or 5′-UTR pGL3 reporter constructs, along with 100ng of pRL-TK Renilla luciferase construct or pRL-null Renilla luciferase construct (Promega) via Lipofectamine 2000 delivery (Life Technologies). Following 24 h transfection, NRVM were either electrically paced at 2 Hz, 30V, 10ms using a Ion Optix myopacer, or treated with, 20ng/mL neuregulin, 200μM phorbol 12-myristate 13-acetateor (PMA), 0.5 μM doxorubicin, 50 μM H₂O₂, or 3 μM blebbistatin for 24hrs.

Cadar A.G., Zhong L., Lin A., Valenzuela M, & Lim C.C. (2014). Upstream Open Reading Frame in 5′-Untranslated Region Reduces Titin mRNA Translational Efficiency. Biochemical and biophysical research communications, 453(1), 185-191.

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