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Comparative Analysis of Dual-Luciferase® Assay Technologies in a High Throughput Microplate Format

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Comparative Analysis of Dual-Luciferase® Assay Technologies in a High Throughput Microplate Format Application Note Luciferase Reporter Gene Comparative Analysis of Dual-Luciferase® Assay Technologies in a High Throughput Microplate Format Peter J. Brescia, Jr. and Peter Banks, BioTek Instruments, Inc., Winooski, VT USA Amy Landreman, Promega Corp., Madison, WI USA Luciferases are commonly incorporated into genetic reporter systems for evaluation of eukaryotic gene expression. Often a combination of two luciferase variants with differing substrate affinities are used in a dual-luciferase assay format. Typically one luciferase acts as an experimental reporter to measure the biological response while the other is a constitutively expressed assay control used to normalize data. Several luciferase variants have been developed exhibiting broad dynamic ranges and excellent sensitivity given the extremely low background signal seen in most experimental systems. Luciferase activity typically proceeds in one of two ways resulting in either a short, intense signal or a longer-lived, more moderate signal referred to as flash or glow type assays, respectively. Several currently available dual-luciferase assay technologies utilizing different luciferase variants were investigated across a range of microplate readers in a high-throughput screening microplate format using automated method. Introduction The importance of luciferase-based assays has and time1. Typically, one of the two reporters’ been well documented over the past several luminescent signal is initiated by substrate decades1. With the advent of genetic engineering addition, measured, and followed by a quenching tools luciferase gene constructs are now routinely reaction and initiation of the second reporter inserted into organisms or transfected into cells signal allowing for a homogeneous assay format. Key Words: where they serve as reporters of gene activity2. This involves generation of a genetic construct High throughput screens often utilize luciferase Dual-luciferase® where the luciferase gene is under the control reporter assays in order to assess effects of Bioluminescence of a transcriptional promoter. Upon activation compounds on target pathways of interest of the target promoter, activity can then be because of automatable reagent addition, NanoLuc® measured. Conversely, when under the control rapid and quantifiable assay read-out, signal Reporter Gene Assay of a constitutively expressed promoter any kinetics compatible with batch processing, and perturbations in luciferase basal activity can compatibility with high throughput microplate DLR™ provide insights into transcriptional control formats. However, scaling luciferase assays Luminescence Assay mechanisms for that gene. These studies into higher-density formats can be challenging can include investigation of receptor activity, due to lower signal intensity and increased risk transcription factor binding, mRNA processing, of variability. In addition, for dual-luciferase protein folding and detection of cellular ATP assays, the quenching required for accurate levels. measurement of the second reporter can be hampered by increased difficulties in achieving In many instances, a dual-luciferase assay format adequate mixing in higher-density microplates. is adopted where one luciferase variant performs Proper quenching depends largely on variables as an experimental reporter and a second as such as plate type, method of reagent dispensing, a control reporter. The experimental reporter and the type and amount of plate shaking. allows correlation of the biological effect being studied while the control serves to normalize Here we investigate the performance of data, eliminating variability due to experimental several luciferase variants and assay formats factors such as transfection efficiency, cell adapted to a high throughput screening BioTek Instruments, Inc. viability and cell number as well as physical (HTS) compatible, 384-well format across P.O. Box 998, Highland Park, conditions such as differences in temperature a range of compatible microplate readers. Winooski, Vermont 05404-0998 USA Phone: 888-451-5171 Outside the USA: 802-655-4740 Email: [email protected] www.biotek.com Copyright © 2016 Application Note Luciferase Reporter Gene Background Recently published work involving luciferase from the deep sea shrimp, Oplophorus, highlighted the development Genetic reporter systems have long been used to of an engineered enzyme and associated substrate with investigate regulatory mechanisms of eukaryotic many of the desirable characteristics mentioned above3. gene expression and cellular physiology in response The engineered luciferase is a stabilized variant of the to environmental stimuli. Reporter constructs require smaller catalytic subunit (Oluc-19) from the heteromeric insertion of the target regulatory element of interest native structure and designated NanoLuc® (NLuc) upstream of a reporter gene which is then incorporated (Figure 2). NLuc, when coupled with substrate, the into the host cell using genetic methods or incorporated coelenterazine analogue furimazine, produces a much into a plasmid and transfected into host cells. A wide brighter light than either Fluc or Rluc with a spectral range of applications have taken advantage of reporter maximum at 454 nm (Figure 3). Additional desired systems. characteristics include excellent compatibility with common cell-based systems3. Bioluminescence, a photon-emitting enzymatic oxidation reaction, occurs in a diverse range of organisms by a generic class of enzymes termed luciferases. Several characteristics of this class of enzymes such as very low background signal and wide dynamic range has made them a popular choice as reporters to investigate cellular physiology1. More recently, luciferase based reagents have been diversified to include quantification of cellular viability, apoptosis and metabolism2,3. Firefly (Photinus pyralis, Fluc) and Renilla (Renilla reniformis, Rluc) luciferases, 61 and 36 kDa, respectively, have been the primary workhorses in luciferase-based applications (Figure 1). Since their introduction, discovery of luciferases from a variety of organisms, primarily marine sources, have been introduced and offer distinct properties such as secretion into cell culture medium and increased Figure 2. Comparison of relative sizes of different luciferases. stability. However, many of the desired properties such as bright luminescence and low background were coupled with either rapid decay or substrate instability or autoluminescence under routine cell-based assay conditions. Additionally, many native luciferase enzymes are found as heteromultimeric complexes ranging in size from 20 kDa to greater than 100 kDa making them too large or difficult to incorporate into cell-based assay systems. Therefore, efforts have been made to simplify the luciferase structure to yield a small, monomeric, stable enzyme with bright, extended light generation capabilities. Figure 3. Bioluminescent reaction catalyzed by NanoLuc luciferase. Dual-luciferase assays measure reporter luciferase activity, typically a control reporter and experimental reporter, sequentially from a single sample. The control reporter allows for normalization of experimental reporter luminescence in a co-transfected, dual-reporter system to account for differences in experimental conditions (Figure 4). Typically this is accomplished by addition of reagent containing substrate specific for a single luciferase species resulting in a stabilized signal. After the signal is Figure 1. Bioluminescent reactions catalyzed by firefly and Renilla read, a second reagent is added to the sample resulting luciferases. in quenching of this reaction while simultaneously initiating the second luciferase reaction. Maximum signal intensity and duration is dependent on individual luciferase reaction kinetics. 2 Application Note Luciferase Reporter Gene reporter activity measured following the appropriate incubation period. For the flash assay integrated injectors in each reader were used for reagent addition followed by 2 second delays before measurements were recorded. For the Glo type assays reagents were dispensed manually using a repeater pipettor. The plates were incubated with shaking on an orbital shaker at room temperature for the recommended incubation period prior to recording measurements. Following the initial read, the second reagent was dispensed and incubated as described above prior to reading. Results Dual-Luciferase® Reporter Assay System Figure 4. Dual Reporter Assay system. Experimental and control reporter vectors are transformed into the target cell. The The Dual-Luciferase® Reporter (DLR™) Assay System experimental reporter expression varies in relation to treatment while the control reporter is constitutively expressed allowing normalization typically uses firefly (Fluc) as the experimental reporter of data between individual samples and experiments. and Renilla (Rluc) as the control. The Fluc signal is measured first followed by Rluc. Both luciferases exhibit “flash” kinetics offering very bright signal and high sensitivity with a short half-life. In this instance, Materials and Methods a simplified system was tested consisting of purified luciferases to illustrate performance with a high level of ® The Dual-Luciferase Reporter Assay System (Cat. No. Fluc expression. Replicate controls consisting of individual ® E1910), Dual-Glo Luciferase Assay System (Cat. No. purified luciferase were added to a 384-well microplate. ® ® E2920), Nano-Glo Dual-Luciferase Assay System (Cat. In
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