Reporter assay and dualluciferaser promega corporation. Single and dualreporter assays cells are inherently complex, and the data available from a single reporter may be insufficient for achieving reliable results. Dual reporter assays like this that use either the same or. Since the introduction of the dualluciferase assay system in the mid1990s, this reporter assay has been widely applied in the study of tfdna interaction 221 222223.
This dualluciferase format allowed detecting compound cytotoxicity or offtarget change in expression during drug screening, therefore improved data quality. Luciferase reporter gene assays are one of the most common methods for monitoring gene activity. This dual luciferase format allowed detecting compound cytotoxicity or offtarget change in expression during drug screening, therefore improved data quality. Thus, dual reporter assays often allow more reliable interpretation of the experimental data by reducing extraneous influences. This system assays cells directly in growth medium. Dualglo luciferase assay kit provides a convenient, rapid, and sensitive procedure for quantifying gene expression.
Improved dualluciferase reporter assays for nuclear receptors. The dualluciferase reporter dlr assay system provides an efficient. In the dlrz assay, the activities of firefly photinus pyralis and renilla renilla reniformis, also known as sea pansy luciferases are measured sequentially from a single sample. 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. Consecutive detection of firefly and renilla luciferase activities in a single sample. Luciferase is an ideal reporter because of the absence of. Transcriptional regulation, coupled to the expression of a luciferase reporter gene, is regularly used to study a wide range of biological events in cultured cells. Dual luciferase reporter dlr assay certification bmg. Dualluciferaser reporter assay system tm046 promega. Luciferase assay was performed with dualluciferase reporter assay system.
The dlr assay is a two step reaction that uses two luciferase enzymes, fire. Dual luciferase assays were performed with the dual luciferase reporter assay system promega corp. Overview of the nanoglo dualluciferase reporter assay. Format of the dualluciferase reporter assay in 96well plates. Superlighttm dual luciferase reporter gene assay kit bioluminescent assay for dual reporter gene systems description the accuracy of reporter assays can be improved by utilizing a dual reporter system. Luciferase reporter assay kit university of michigan. 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. The luciferase reporter assay is commonly used as a tool to study gene expression at the transcriptional level. Luciferase reporter technology gives the researcher unparalleled sensitivity, dynamic range, versatility and ease of use when. Detect dual luciferase expression on the flexstation 3. Dual reporter assay luciferase is widely used as a reporter due to its sensitivity and linear range, and a wide variety of luciferasebased systems are available for. Using the synergyht multidetection microplate reader to.
In the dlr assay, the activities of firefly photinus pyralis and renilla renilla reniformis, also known as sea pansy luciferases are measured sequentially from a single sample. Application of the dualluciferase reporter assay to the analysis of. Dualreporter assays enable researchers to obtain additional information from complex systems with minimal effort. Combined with choices for traditional firefly and renilla luciferase assay kits to provide compatibility with multiple systems.
Flexstation 3, a nextgeneration multimode microplate reader, with. In the nanoglo dual luciferase reporter assay, firefly luciferase activity is measured first using the supplied oneglo ex luciferase assay reagent. Dual luciferase gene reporter assays to study mirna. Assay procedure for 96well plates using dualsample injection. The dualluciferase reporter dlr assay system provides an efficient means of performing. The dual luciferase reporter dlr assay system provides an efficient means of performing dual reporter assays. Pdf dual luminescencebased reporter gene assay for. Dualluciferase assays in 96 and 384well formats on the new spectramax l by cathy olsen, ph. Because of their sensitivity, dynamic range, and lack of endogenous activity, luciferase assays have been particularly useful for functional genomics in cellbased assays, such as rnai screening. Detection of luciferase reporter assays bmg labtech. Passive lysis buffer using either the passive or active lysis procedure. However, cypridina and gaussia luciferases are secreted. 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. Performing most dualluciferase assays involves adding two reagents to each sample and measuring luminescence following each addition.
In the dlr assay, the activities of firefly and renilla luciferases are measured sequentially from a. Dualluciferase reporter assay system technical manualpdf. The firefly luciferase reporter is measured first by adding luciferase assay reagent ii. In the dlr reporter assay, the activities of firefly photinus pyralis and renilla renilla reniformis, also known as sea pansy luciferases are measured sequentially from a single sample. The firefly luciferase and the renilla sea pansy luciferase can discriminate between their respective bioluminescence substrates.
The result is an excellent tool for monitoring gene expression in cells due to wide dynamic range and sensitive detection. All reagents were prepared as described by the manufacturer. A new dual luciferase assay using nanoluc enables a. Dual luciferase reporter system promega 24 well plate transfections cells should be transfected with a test reporter firefly luciferase and a normalization reporter driven by a constitutive promoter renilla luciferase prltk thymidine kinase promoter works. 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 increased brightness of nluc and improved fluc inhibition. Since firefly and renilla luciferases use different substrates, both can be measured in the same well using a dual luciferase detection assay. It has broad applications across various fields of cell and molecular biology wherever you want to measure or track expression of a cloned gene. Reagents can be used with injectionbased protocols or with cell lysates made with passive lysis buffer.
The dualluciferase reporter dlr assay system provides an efficient means of performing two reporter assays. Dual luciferase assay system for rapid assessment of gene. It is widely used because it is convenient, relatively inexpensive, and gives quantitative measurements instantaneously. The dual luciferase reporter dlr assay systemac provides an efficient means of performing dual reporter assays. Get at least 100 assay points from a 100 assay point kit. 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. The dual luciferase reporter dlr assay systema,b provides an efficient means of performing dual reporter assays. Introduction reporter gene assays are used to study the expression of eukaryotic genes.
Four different mammalian cell types were cotransfected with firefly and renilla luciferase. The activity of renilla reniformis luciferase was determined and normalized with that of firefly luciferase, and detected with a dualluciferase reporter assay system. A second luminescent reporter, renilla luciferase, is often used under the control of a constitutive promoter to normalize for sources of variability such as transfection efficiency and cell number. Dualreporter assay using two secreted luciferase genes. 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. Dualluciferase reporter assay system technical manualpdf 771 kb. Dualluciferase reporter assay system from promega utilizes differing luminescent properties of firefly and renilla luciferase to normalize activity of an experimental reporter with an internal control. Luciferase reporter assay in drosophila and mammalian. Protocol for dna transfection and luciferase assay using pfrwt plasmid. Hidden camera investigation on what really happens to your car cbc marketplace duration. In dual reporter gene assays cells are transfected with two vectors, the first containing an experimental reporter gene coupled to a regulated promoter.
The nanoglo dualluciferase reporter nanodlr assay system is a homogeneous reagent system that allows you to sequentially detect the activities of firefly photinus pyralis luciferase and nanoluc luciferase nluc from a single sample. Protocol for dna transfection and luciferase assay using. Genecopoeias secretepair system offers the unique advantage of a secreted dualreporter luminescence system, which allows reporter assays in live cells. Most experimental reporter constructs drive firefly luciferase, whereas renilla. Luciferasebased reporter assays employ insertion of a genetic regulatory element upstream of the luciferase gene. Grow li cells transformed with pfrwt plasmid dna in appropriate volume of lb or 2xyt media with ampicillin overnight and isolate dna using standard qiagen mini or maxiprep protocol. Dualluciferase reporter assay system promega corporation. Dualluciferase assays in 96 and 384well formats on the. 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. A nextgeneration dualluciferase reporter assay using. In mammalian cells, dual reporters have been commonly used to improve the accuracy of transientreporter assays when analyzing gene expression. Does not apply to renilla luciferase substrate renlite substrate, whose long term storage is 20 c. A noncommercial dual luciferase enzyme assay system for reporter gene analysis. Resuspend the lyophilized luciferase assay substrate in luciferase assay buffer ii 10ml for cat.
These luciferase reporter assays provided better research and drug discovery tools for studying the functions of full length nuclear receptors and ligand binding domains. Cellbased assays validation and comparison of singlestep. The firefly luciferase reagent is first added to the cells in medium directly. The luciferase reporter assay kit is suitable for use with any standard transfection experiment utilizing firefly luciferase as a reporter. The absorbance of oxidized tmpd is measured spectrophotometrically at 610 nm. Promega manufacturing and delivery systems continue to be fully operational during the covid19 outbreak. One of the reporter genes is correlated with the promoter. A veritas microplate luminometer method for promega. Dualluciferase reporter assay system harvard university. Pdf a noncommercial dual luciferase enzyme assay system. The firefly luciferase reporter assay provides rapid results of cellular transfections 14. Rna interference rnai is a mechanism that inhibits gene.
152 1264 1520 1404 716 771 1249 1650 736 1332 1040 6 950 333 1110 620 473 1026 774 1103 105 1266 153 884 611 1618 96 1278 183 346 778 1362 194 1175 1297 387 1432 330 652 316 1214 1342 963