Dual-glo™ luciferase assay system: a homogeneous dual-reporter system

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DUAL-GLO™ LUCIFERASE ASSAY SYSTEM:
the extraction of useful data by differentiating genetic A HOMOGENEOUS DUAL-REPORTER SYSTEM
responses of interest from non-relevant influences. Suchinfluences may include “edge effect” in multiwell plates, by Erika Hawkins, M.Sc., Braeden Butler, B.S., Keith Wood, transfection efficiency in transiently transfected cells, and Ph.D., Michael O’Grady, M.S., Laurie Orr, B.S., and Michael other sources of interference, often recognized simply as Abstract
Interference in the reporter response can also arise when Promega has developed a new, homogeneous reporter underlying genetic events are masked by physiological assay, the Dual-Glo™ Luciferase Assay System, for factors such as cell viability. This is problematic for monitoring both firefly and Renilla luciferases distinguishing genetic downregulation from cytotoxicity, expressed in mammalian cells in 96- and 384-well particularly when using specific downregulation as a means plates. The new assay system integrates cell lysis and of identifying novel receptor antagonists, because a reduction luminescence chemistry, and the luminescence kinetics of reporter expression can be confused with cytotoxicity.
have been extended over several hours. The Distinguishing Downregulation from Cytotoxicity
homogeneous assay format allows dual-reporter assaysto be more easily and rapidly performed by reducing To model this circumstance, single- and dual-reporter sample processing requirements and eliminating the measurements were made in cells expressing Renilla need for reagent injectors in luminometers. luciferase under control of the Tet-Off promoter and fireflyluciferase under control of the CMV promoter (Figure 1).
Introduction
Reporter activity was measured after adding titrated Although reporter genes are widely used for rapid amounts of doxycycline or G418 antibiotic to the cells.
evaluation of cellular physiology (1,2), a single reporter Doxycycline is expected to specifically downregulate the may not convey sufficient information for reliable Renilla expression coupled to the Tet-Off promoter, while interpretation of the experimental data. For this reason, G418 is expected to kill the cells. Because both compounds dual reporters are commonly used, most notably the reduce Renilla luminescence with increasing dose, it is not bioluminescent firefly and Renilla luciferase genes.
possible to distinguish specific genetic regulation from cell Promega recently introduced the Dual-Glo™ Luciferase death using a single reporter. This is demonstrated in Panel Assay System(a,b,c) (3) for dual reporter measurements in A, where only Renilla luciferase activity was assayed. In a homogeneous assay format. Like Promega’s Dual- contrast, by using the firefly luciferase as an internal Luciferase® Reporter Assay System(a,b,c), the new Dual- reference, the distinction between genetic response and Glo™ Assay allows sequential measurement of both firefly cell death is clear. Doxycycline reduces only Renilla and Renilla luciferases from one sample. However, the luminescence, but cell death caused by G418 reduces the kinetics of the luminescent reactions have been extended luminescence of both reporters. This distinction can be to allow for processing multiple samples before initiating readily displayed as the ratio of Renilla to firefly measurements. Reagent can be added to all samples in a luminescence. In Panel B, the Renilla luciferase activity is multiwell plate, or even to a stack of multiwell plates, normalized to the co-transfected firefly luciferase control.
before placing the plates in a luminometer. To furthersimplify sample processing, the lytic components of the The Dual-Glo™ Assay can be performed by assay have been combined with the luminescent chemistry to yield an integrated assay formulation. Consequently, the Dual-Glo™ Assay can be performed by adding thereagents directly to cells in culture medium and measuring Experimental strategies involving dual reporters havebecome increasingly common, preferred for their ability to Reporter genes offer an excellent means for studying provide reliable and meaningful data. Reporter analyses also complex genetic regulatory networks. However, this are increasingly being performed in multiwell plates. The complexity can also make it difficult to isolate and culmination of these trends is high-throughput screening, characterize a specific physiological pathway without where huge numbers of samples are quantitatively analyzed.
interference from other elements within the system. The The Dual-Glo™ Assay System was developed to support significance of this interference on reporter responses can these requirements by providing a simple, homogeneous be realized only through a properly configured reference, means of quantifying both the firefly and Renilla luciferases typically from a secondary reporter. Dual reporters facilitate from mammalian cells in culture medium.
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Doxycycline
Luminescence
Renilla
Relative
[Inhibitor] (µg/ml)
Doxycycline
Relative Response Ratio
[Inhibitor] (µg/ml)
Figure 1. Differentiating genetic downregulation from cytotoxicity. CHO cells were transiently transfected with Renilla luciferase
under the control of the Tet-Off promoter and firefly luciferase under the control of the CMV promoter. Cells were titrated with a
specific inhibitor of Renilla luciferase expression (doxycycline) or a cytotoxic agent (G418 antibiotic). Panel A illustrates that the
output from a single reporter is similar under both inhibitors, because both test compounds yield diminished reporter
luminescence. The y-axis shows relative Renilla luminescence as a percent of sample without inhibitor. In contrast, Panel B shows
that a dual-reporter system can clearly distinguish between specific downregulation of the gene and cytotoxicity. The output is
recorded as a relative response ratio (RRR), where the sample, negative and positive controls are reported as the ratio of Renilla
luminescence to firefly luminescence. Measurements of the relative response ratios were also more precise than the
measurements of a single reporter (average relative standard deviation of 6.5% in Panel B compared with 13% in Panel A). RRR=
[sample – negative control] / [positive control – negative control], as a percent.
References
Ordering Information
Product
1. Alam, J. and Cook, J.L. (1990) Reporter genes: Application to the study of mammalian gene transcription. Anal. Biochem. 188, 245–54.
2. Wood, K.V. (1991) In: Bioluminescence and Chemiluminescence: Current Status, Stanley, P. and Kricka, L., eds. John Wiley and (a)Certain applications of this product may require licenses from others.
3. Hawkins, E. et al. (2002) Dual-Glo™ Luciferase Assay System: (b)U.S. Pat. Nos. 5,283,179, 5,641,641, 5,650,289, 5,814,471, Australian Pat. No. 649289 Convenient dual reporter measurements in 96- and 384-well and European Pat. No. 0 553 234 have been issued to Promega Corporation for a fireflyluciferase assay method, which affords greater light output with improved kinetics as plates Promega Notes 81, 22–26.
compared to the conventional assay. Other patents are pending.
(c)U.S. Pat. No. 5,744,320 and Australian Pat. No. 721172 have been issued to Promega Protocols
Corporation for quenching reagents and assays for enzyme-mediated luminescence. Otherpatents are pending.
Dual-Glo™ Luciferase Assay System Technical Manual Dual-Glo is a trademark of Promega Corporation. Dual-Luciferase is a trademark of Promega Corporation and is registered with the U.S. Patent and Trademark Office.
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