A commonly used reporter gene is the luciferase gene from the firefly. Development of reporter assays to measure regulation of gene expression will facilitate understanding of regulatory mechanisms involved in this process. The dual reporter luciferase method is well suited for studying gpcr modulators in a highthroughput format. Performing the nanoglo dual luciferase reporter assay 3. The system, provided with cell culture lysis reagent cclr, also can be used for reporter quantitation.
Therefore, understanding the mechanisms by which reporter enzyme activity can be modulated by small molecules is critical to the interpretation of hts data. In rare cases, this activity appears counterintuitive. What is the reason for the effect on renilla signal in the. Learn how luciferase can be used in concert with fluorecent proteins in nano lanterns. Luciferase assays have since become a gold standard in gene expression analysis and a luciferase gene one of many available to choose from is now a common feature in reporter plasmids.
In this protocol, cells transfected with a luciferase reporter plasmid are lysed using a. A specific mechanism for nonspecific activation in. This bioassay record aid 411 belongs to the assay project probe summary for inhibitors and stabilizers of firefly luciferase, which is associated with the summary aid 2309 and a total of 18 additional bioassay records in pubchem. Using a model system of exogenous or endogenous receptors in hek293 cells, we demonstrated that all four major g protein subfamilies and downstream pathways can be studied in one luciferase reporter assay format. The protocol is written to accommodate a 96well plate, but this can just as easily be. M mir26a mimic or negative control mimic with 500 ng of psicheck2 reporter plasmids using lipofectamine 3000 thermo fisher scientific and performing the dual luciferase reporter assay system promega after 48 hours. These cells either constitutively or inducibly express a reporter gene such as gfp, rfp, luciferase, or lacz. Therenilla luciferase assay has a linear range covering six orders of magnitude and allows for the detection of approximately. The name was first used by raphael dubois who invented the words luciferin and luciferase, for the substrate and enzyme, respectively. The improved reporter assays showed larger assay dynamics in shorter assay time than using regular luciferase. May 16, 2018 dual reporter assay luciferase is widely used as a reporter due to its sensitivity and linear range, and a wide variety of luciferase based systems are available for monitoring promoter activity in. Luciferase genes vary in stability, light output and spectral characteristics, and reporter kits are available with different luciferases for both single and dualspectral analyses. The firefly luciferase complementation assay is widely used as a bioluminescent reporter technology to detect proteinprotein interactions in vitro, in cellulo, and in vivo.
The dualluciferase reporter dlr assay and dualglo assay enable the sequential measurement of both firefly and renillaluciferases from one sample. In this protocol, cells transfected with a luciferase reporter plasmid are lysed using a detergentcontaining buffer. The dual luciferase reporter dlr assay system provides an efficient means of performing two reporter assays. Transfection basics, principle, mechanism and optimization of mammalian cell. Due to the longlasting signals, the luciferase assay, on the other hand, provides the opportunity to take multiple measurements over a longer time period. Luciferase reporter assays allow you to investigate promoter activity by measuring light output from luciferase enzyme that is expressed under the control of your promoter of interest. Previously, we have shown that the luciferase reporter vectors pgl4 series containing minimal promoter and destabilized luciferase gene improve the responsiveness of camp response element to g s coupled receptor 8 fan f, wood kv. Luciferase assay was performed with dualluciferase reporter assay system kit.
Rna interference rnai is a mechanism that inhibits gene expression before a gene. Reporter protein assays use colorimetric, radioactive or luminescent methods to measure enzyme activity present in a cell lysate. The dlr and dualglo assays provide rapid and convenient means for achieving greater control over the biological significance of reporter data by differentiating genetic responses of. Upon the interaction of a protein pair, complemented firefly luciferase emits light through the adenylation and oxidation of. The cell contains a firefly luciferase gene under the control of are antioxidant response element stably integrated into hep g2 cells. Using luciferase reporter assays to screen for gpcr modulators. Luciferase reporter assays were performed by cotransfecting hek293 cells with 8. The polya tail length was analyzed by rnase h digestion and northern blotting as previously described. Highthroughput screening hts assays used in drug discovery frequently use reporter enzymes such as firefly luciferase fluc as indicators of target activity.
Typically, a promoter region containing 2 to 3 kb of a 5. Luciferase reporter assay system for deciphering gpcr. Bioglo assay reagent contains a new luciferase substrate, resulting in a reagent that is more stable and more tolerant to sample components than. Reporterlabeled cell lines are useful for monitoring various biological functions. A protocol for reporter quantitation in mammalian cells. It has broad applications across various fields of cell and molecular biology wherever you want to measure or track expression of a cloned gene. Because of the requirement of atp, firefly luciferases have been used extensively in biotechnology.
Pdf detection of e2finduced transcriptional activity using. Luciferase assay system protocol promega corporation. Luciferase reporter mice for in vivo monitoring and ex. Aid 411 qhts assay for inhibitors of firefly luciferase. Choose your luciferase reporter gene firefly luciferase or renilla. The sensitivity was adjusted as necessary to ensure light levels remained within the instruments detection limits. An important caveat to consider, however, is that compounds can directly affect the reporter, leading to nonspecific but highly reproducible assay signal modulation. Improved assay performance by using destabilized luciferase reporter. Detection of e2finduced transcriptional activity using a dual luciferase reporter assay chapter pdf available in methods in molecular biology clifton, n. The firefly luciferase reporter is measured first by adding luciferase assay reagent ii lar ii to generate a stabilized luminescent signal. Dual reporter assays like this that use either the same or. Engineered luciferase reporter from a deep sea shrimp. Mar 03, 2009 highthroughput screening hts assays used in drug discovery frequently use reporter enzymes such as firefly luciferase fluc as indicators of target activity.
The firefly luciferase reporter assay provides rapid results of cellular transfections 14. The firefly luciferase reporter assay is initiated by adding an aliquot of lysate to luciferase assay reagent ii. Transient transfection and luciferase assay 20121212 youtube. The firefly luciferase reagent is first added to the cells in medium directly. Viafect transfection reagent transfection assay e4981.
Then, rip was performed to assess the combination between mir1443p and linc00461 or kpna2, and dual luciferase reporter assay was used to validate the targeted relationship between mir1443p and kpna2. It is widely used because it is convenient, relatively inexpensive, and gives quantitative measurements instantaneously. Mechanism of ptc124 activity in cellbased luciferase. Transient transfection and luciferase assay 20121212. General considerations the nanoglo dualluciferase reporter assay system is designed to be used with many media types and has been validated for use with the following culture media containing 010% serum. We show that although ptc124 and related compounds cause apparent activation in cellbased nonsense codon suppression assays using a fluc reporter, they are inactive in the same assay when a different bioluminescent reporter, renilla reniformis luciferase rluc is used as the reporter. An androgen response element driven reporter assay for the. The importance of bioluminescence in enabling a broad range of highthroughput screening hts assay formats is evidenced by widespread use in industry and academia. Dualreporter assay using two secreted luciferase genes. The bioglo luciferase assay system provides a highly sensitive, robust, homogeneous reagent for the detection of firefly luciferase reporter gene expression in the adcc reporter bioassay. An important caveat to consider, however, is that compounds can directly affect the reporter, leading to nonspecific but highly reproducible assay. The 293ft producer cells were grown in white walled, flat surface 96well plates and were cotransfected with ar expression vector and are reporter vectors according to the manufacturers instruction invitrogen, cat. Mar 03, 2009 firefly luciferase is an exquisitely sensitive reporter with a halflife of.
In vitro transcribed rnabased luciferase reporter assay. Here we describe a protocol to study mrna translation using an in vitro transcribed rnabased luciferase reporter assay in poxvirusinfected cells. Both words are derived from the latin word lucifer meaning lightbringer. Because transfection efficiency in such assays can be low, promoters are commonly fused to heterologous reporter genes that encode enzymes that can be quantified using highly sensitive assays. It involves classical luciferase protocol in which the chimeric plasmid luciferase gene and the target promoter is transfected into the cells. The pheromoneresponsive luciferase reporter assay for rgs4 inhibitors was conducted as a singledose assay at a final compound concentration of approximately.
Mathematical model of the firefly luciferase complementation. The luciferase assay is a very sensitive and convenient way to examine the transcriptional activity of a gene. Identification of a novel circadian clock modulator. Aug 26, 20 the luciferase reporter assay is commonly used as a tool to study gene expression at the transcriptional level. Luciferase mrna transfection of antigen presenting cells. Reporter labeled cell lines are useful for monitoring various biological functions.
Although it has been suggested that kinetics of light production in the. Firefly luciferase is an exquisitely sensitive reporter with a halflife of. Dualluciferase reporter assay system harvard university. Over 600,000 random and chemically diverse small molecule compounds were screened.
It has broad applications across various fields of cell and molecular biology wherever you want to measure or. Reporter constructs rely on the insertion of a genetic regulatory. Indigos aryl hydrocarbon receptor ahr assay is a cellbased genetic reporter assay that utilizes the luciferase reporter gene aryl hydrocarbon receptor agonist and antagonist assays functionally linked to an ahrresponsive promoter. To assay the samples, 10 al of the cell lysate were added to 100 al of the luciferase assay buffer, swirled briefly, and placed in the luminometer. Mechanism of ptc124 activity in cellbased luciferase assays of nonsense codon suppression douglas s. Nanoglo dualluciferase reporter assay nanodlr system. Luciferase reporter mice for in vivo monitoring and ex vivo. Nov 08, 2019 all you wanted to know about luciferase but were afraid to ask using luciferase experimentally. Firefly luciferase is the lightemitting enzyme responsible for the bioluminescence of fireflies and click beetles. Promega manufacturing and delivery systems continue to be fully operational during the covid19 outbreak. Arebinding protein zfp36l1 interacts with cnot1 to. Reagents for luciferase reporter assays thermo scientific turbofect transfection reagent deliver dna plasmids and expression vectors into eukaryotic cells with this highly efficient, easytouse, nonimmunogenic transfection reagent. Upon the interaction of a protein pair, complemented firefly luciferase emits light through the adenylation and oxidation of its substrate, luciferin.
Reporter gene assays are an important tool for monitoring cellular events associated with gene expression, gene regulation, and signal transduction. Transfection guide overview of transfection methods. Our teams are in regular contact with suppliers and distributors worldwide and are taking all steps necessary to address both demands for diagnostic tools and reliable delivery of all products as quickly as possible. When a transient or stable transfection assay is developed for a promoter, a primary objective is to quantify promoter strength. Firefly luciferase, one of the most commonly used reporters, produces light by catalyzing a bioluminescence reaction that oxidizes dluciferin in the presence of oxygen and atp. Luciferase is a generic term for the class of oxidative enzymes that produce bioluminescence, and is usually distinguished from a photoprotein. The luciferase reporter assay was performed by nanoglo luciferase assay systems promega. Luciferase reporter gene assay dualspectral luciferase dual reporter luminescence renilla gaussia firefly introduction reporter assays have long been used to investigate regulatory mechanisms of gene expression in response to environmental stimuli in a variety of organisms. Atcc is continually accessioning and creating new reporter cell lines to quicken the pace of your research.
The glow is widely used as an assay for luc expression, which acts as a reporter for the activity of any genetic regulation that controls the expression of its gene. This animation describes the basic concept of reporter assays using luciferase, and explains how a dualluciferase assay works. Mechanism of ptc124 activity in cellbased luciferase assays of nonsense codon suppression. For example, renilla luciferase is not secreted, and thus the pierce renillafirefly luciferase dual reporter assay kit will not work for this type of timecourse assay.
What is the reason for the effect on renilla signal in the luciferase assay and what is the mechanism of transcription without promoter. Signosis has developed p53 luciferase reporter hela stable cell line, in which luciferase activity is specifically associated with the activity of p53. The dual luciferase fireflyrenilla assay system is designed to be used for highthroughput, rapid quantitation of both firefly and renilla luciferases from a single sample in mammalian cell culture. We exploited this stressinduced splicing mechanism to generate a lentivirus vector containing a stressinducible gaussia luciferase reporter plvcmvxbpglucbcpuro fig. Reporter genes are essential tools in the study of gene expression and regulation, as they are designed to function as surrogates for genes involved in key cellular processes and disease. Luciferase reporter assay kit university of michigan. Kinetic analysis and modeling of firefly luciferase as a.
The luciferase reporter assay kit is suitable for use with any standard transfection experiment utilizing firefly luciferase as a reporter. The dualluciferase reporter dlr assay system contains two different luciferase reporter enzymes that are expressed simultaneously in each cell. Aryl hydrocarbon receptor assay ahr indigo biosciences. Dualspectral luciferase reporter assays using luciferase enzymes with nonoverlapping luminescence spectra can be measured simultaneously by filterbased detection in a single sample using a single reagent. Allard and kevin kopish, promega corporation luciferase reporter technology gives the researcher unparalleled sensitivity, dynamic range, versatility and ease of use when investigating questions that involve gene regulation. Dlrz assay system, the linear range of the firefly luciferase assay is seven orders of magnitude, providing detection sensitivity of. In the dlr assay, the activities of firefly and renilla luciferases are measured sequentially from a single sample. The luciferase assay kit is intended for the detection of the expression of firefly luciferase and is not suitable for the assay of luciferase from other origins. Dec 12, 2012 transient transfection and luciferase assay 20121212. Application noteluciferase assay and bio tek synergy microplate readers. A bioluminescence reporter assay was developed that expresses firefly luciferase under the control of the glur1 5.
Luciferase is particularly useful as a reporter in living cells and organisms. All you wanted to know about luciferase but were afraid to ask using luciferase experimentally. Luciferase reporter assays to determine yaptaz activity. A further advantage of using geneencoding rna is that, together with the luciferase reporter gene ivt rna, any other antigen or vaccine ivt rna of interest can be cotransfected. For the conventional luciferase reporter assay, each indicated luciferase reporter and expression construct was cotransfected together with 400 ng of a renilla luciferase reporter construct prlbasic, as an internal control for transfection efficiency, into nih3t3 cells using lipofectamine plus reagent invitrogen.
Luciferase cell culture lysis reagent promega, madison, wi, sonicated and vortexed, followed by centrifugation for 10 minutes at 4c and 15,000 rpm. A wide variety of luciferase enzymes have been discovered, although the general mechanism of the lightproducing reaction for all of the enzymes is the oxidative decarboxylation of the luciferase substrate in the presence of o 2 to yield photon emission light. The firefly luciferase and the renilla sea pansy luciferase can discriminate between their respective bioluminescence substrates and do not crossactivate. Jan 27, 2015 the luciferase reporter gene assay, based on the responsibleelementregulated expression of luciferase, has been widely applied in the highthroughput functional assay for many targets because of. Luciferasebased genetic reporter assays provide sensitive methods for. Spectramax duoluc reporter assay kit molecular devices. Furthermore, transwell was employed for the examination of cell invasion and migration in breast cancer. Dec 10, 2018 bioluminescent reporter assay for monitoring er stress in human beta cells. Quantitation of luminescent signal from each of the luciferase reporter enzymes may be performed immediately following lysate preparation without the need for dividing samples or performing additional treatments. A bioluminescence reporter assay for retinoic acid control of. Maguire, and james inglese2 nih chemical genomics center, national institutes of health, bethesda, md 208923370.
However, cypridina and gaussia luciferases are secreted. Therefore, the cell line can be used as a reporter system for monitoring the activation of p53 triggered by stimuli treatment, enforced gene. Bioluminescent reporter assay for monitoring er stress in. Use of luciferase probes to measure atp in living cells. Assays based on luminescence are very popular for several reasons. In addition, we present a detailed protocol for the in vivo detection of extracellular atp in mice using luciferase transfected reporter cells. To determine if a protein is able to activate or suppress transcription of a gene of interest, a scientist uses recombinant dna technology to produce a construct in which the genes promoter is placed adjacent to a luciferase reporter gene. The are reporter hep g2 cell line is designed to monitor nrf2 antioxidant response pathway. The enzyme catalyses the oxidation of firefly luciferin, requiring oxygen and atp. Mar 10, 2017 luciferase reporter gene assays are a type of reporter gene assay that utilizes the light generated by one of a family of enzymes known as luciferases. The luciferase reporter assay is commonly used as a tool to study gene expression at the transcriptional level. Mechanism of ptc124 activity in cellbased luciferase assays. Cellbased assays validation and comparison of singlestep.