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Veritas™
Microplate Luminometer Method for Promega Dual-Luciferase® Reporter (DLR) Assay 1. INTRODUCTION The Turner BioSystems Veritas™ Microplate Luminometer in combination with Promega's Dual-Luciferase® Reporter (DLR) Assay kit provides a convenient, rapid, and sensitive procedure for quantifying gene expression. 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. Luciferase is an ideal reporter because of the absence of endogenous luciferase activity in mammalian cells, and the functional enzyme is created immediately upon translation1,2. The Dual-Luciferase® Reporter
(DLR) Assay System contains two different luciferase reporter enzymes
that are expressed simultaneously in each cell. Typically, the experimental
reporter is correlated with the effect of specific experimental conditions,
while the activity of the co-transfected "control" reporter gene provides
an internal control, which serves as the baseline response. Normalizing
the experimental reporter gene to the activity of an internal control
minimizes the variability caused by differences in cell viability and
transfection efficiency. Thus, dual reporter assays allow more reliable
interpretation of the experimental data by reducing extraneous influences.
The experimental and control luciferase enzymes used in the Dual-Luciferase® Reporter (DLR) Assay have distinct evolutionary origins. The firefly luciferase
and the Renilla (sea pansy) luciferase can discriminate between
their respective bioluminescent substrates and do not cross-activate. The Veritas™ Microplate Luminometer
can detect as little as 1X10-19 moles firefly luciferase enzyme
using Luciferase Assay Reagent II (LAR II) and
2. MATERIALS REQUIRED
3. EXPERIMENT PROTOCOL 3.1 Reagent Preparation Luciferase Assay Buffer II and Luciferase Assay Substrate: Use as supplied. Store at -20°C, where it is stable for up to 6 months. The Luciferase Assay Substrate may also be stored at 4°C for up to one month. Transfer the contents of one bottle of Luciferase Assay Buffer II into one vial of Luciferase Assay Substrate. Mix by inversion until the substrate is thoroughly dissolved. Use reconstituted Luciferase Assay II Reagent (LAR II) on the same day it is prepared, or aliquot into working volume and store at -20°C for 1 month or 70°C for up to one year. Stop & Glo® Substrate and Stop & Glo® Buffer: Use as supplied. Store below Stop & Glo® Buffer Substrate Solvent: Use as supplied. Store below 25°C. To make Stop & Glo® Reagent, dilute the 50x Stop & Glo® Substrate to 1x concentration using Stop & Glo® Buffer in a glass or siliconized polypropylene tube. Mix by inversion. Use reconstituted Stop & Glo® Substrate on the same day it is prepared or store at -20°C for up to two weeks. Passive Lysis Buffer: To make 1x Passive Lysis Buffer, dilute the 5x Passive Lysis Buffer in DI water. Store below 25°C. Note: The temperature of the Luciferase Assay Buffer II and Stop & Glo® Buffer should be held constant at room temperature while quantifying luminescence since luciferase activity is temperature dependent. Reagent stored frozen after reconstitution must be thawed below 25°C to ensure reagent performance. Mix well after thawing. The simplest method for thawing is placing the reagent in a water bath at room temperature. 3.2 . Instrument Setup 3.2.1 Double click on the Veritas™ icon to start the software. 3.2.2 Click on "Run Promega Protocol" from the "Welcome to Veritas" dialog box. 3.2.3 Browse the files in the "DLR" folder
and choose the appropriate protocol. For example, if you are using DLR
with one injector select "DLR with one injection." 3.2.5 Enter your information into the "Experiment", "Operator", "Plate No.", and "Notes" fields in the "Main Dialog Box". 3.3 Sample Analysis 3.3.1 Prepare the 96-well plate containing
lysed cell cultures. 3.3.2 Prepare the injectors. Place the
intake tubing for injector 1 into the bottle of LAR II. Place the intake
tubing for injector 2 into the bottle of Stop & Glo® Reagent. Prime both injectors using the "Prime" tab located on the "Main
Dialog Box". 3.3.3 Insert the plate into the Veritas™ Microplate Luminometer and click "Start" to begin assay. RLU values measured by the Veritas™ Microplate Luminometer will appear in the Excel spreadsheet after all the selected wells in each row have been read. If you encounter an error message, refer to the troubleshooting guide for more information. 3.3.4 Once the measurements are complete you can access Excel to analyze your data. 3.3.5 Remove your plate after the measurements are complete. You can choose the "Reverse Purge" tab to return unused reagent to the bottle. Be sure to flush injectors thoroughly after use. 4. REFERENCES 1. Ow, D.W. et al. (1986) Transient and stable expression of the firefly luciferase gene in plant cells and transgenic plants. Science 234, 856—9. 2. De Wet, J.R. et al. (1987) Firefly luciferase gene: structure and expression in mammalian cells, Mol. Cell. Biol. 7, 725—37. 5. ABOUT PROMEGA CORPORATION
6. ABOUT TURNER BIOSYSTEMS Phone: (408)
636-2400 Contact us
via our contact
form Mailing Address: CAUTION: The lyophilized Luciferase® Assay Substrate contains dithiothreitol (DTT) and is therefore classified as hazardous. The reconstituted reagent is not known to present any hazards as the concentration of DTT is less than 1%. However, we recommend the use of gloves, lab coats and eye protection when working with these or any chemical reagents. Promega and Turner BioSystems assume no liability for damage resulting from handling or contact with these products.
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