The accuracy of reporter assays can be improved by utilizing a dual reporter system. One of the reporter genes is correlated with the promoter of interest and is used to assess the effects of specific experimental conditions, while the second reporter is used as a control and serves as a baseline response. The Dual-Luciferase Reporter Gene Assay allows for the sequential measurement of the activity of two different luciferases, firefly (FFL) and Renilla (RL), in a single sample. The firefly luciferase luminescence is measured first by addition of the FFL Reagent. Next, the RL Reagent is added to the same well. The RL Reagent simultaneously quenches the firefly luciferase luminescence and initiates the Renilla luciferase reaction. The light production of both reactions can be conveniently measured on a luminometer. Reaction schemes:ATP+D-luciferin+O2 oxyluciferin+AMP+PPi+CO2+light Firefly Luciferase Coelenterazine+O2 Coelenteramide+CO2+lightRenilla Luciferase This bioluminescent dual reporter gene assay is extremely sensitive and is especially suitable for quantifying dual luciferase expression in recombinant cells or in cell free transcription/translation reactions. Assays can be performed in tubes, cuvettes or multi-well plates.
Key Features:High sensitivity and wide detection range: detection of as little of 2fg luciferase
Compatible with routine laboratory and HTS formats: assays can be performed in tubes or microplates, and measured with any luminometer. Can be readily automated on HTS liquid handling systems
Fast and convenient: three step assay allows detection of dual luciferase levels within 20 minutes.
Applications:Gene Regulation: gene expression level, characterization of promoter activity, modulation of gene expression by receptors, transcription factors and small molecules
Drug Discovery: high-throughput screen for gene modulators.
Kit Contents: (100 Assays In 96-Well Plates)Lysis Buffer: 25ml FFL Reagent: 25 mgAssay Buffer: 25ml RL Reagent: 400 mgCustom bulk reagent available upon request
Storage conditions. Store all Reagents at -20°C. Shelf life: 12 months after receipt
Precautions: reagents are for research use only. Normal precautions for laboratory reagents should be exercised while using the reagents. Please refer to Material Safety Data Sheet for detailed information.
Procedures:Reagent Preparation: fresh reconstitution of the FFL and RL Reagents in Assay Buffer is recommended. Calculate the amount of each reagent needed (100ul of each per assay for 96 well plates or 25ul per assay for 384 well plates) and weigh out sufficient amounts of each reagent (2 mg/mL for FFL Reagent and 35 mg/mL for RL Reagent). Gently shake each reconstituted reagent until they are completely dissolved (~10 min). The reconstituted FFL Reagent is stable for up to 4 weeks when stored at -20 °C; however, prolonged storage of reconstituted RL Reagent is not recommended and ideally it should be used within 4 hours.
Sample Preparation:1. Remove media from cell cultures and wash cells with PBS
2. Add enough Lysis Buffer to fully cover the cell monolayer (e.g. 50ul per well of a 96 well plate) and place culture plates on an orbital shaker. Gently shake at RT for 15 min
3. Transfer lysates to tubes for storage (lysates can be stored for 8 hours on ice or overnight at -20°C). Alternatively, the dual luciferase assay can be performed directly in the culture plate if cells were cultured in opaque plates
Dual Luciferase Assay Procedure:1. If not assaying directly in the culture plate, transfer lysate to an opaque white plate (50ul for 96 well plates, 12.5ul for 384 well plates)
2. Add reconstituted FFL Reagent to each well. (100ul for 96 well plates, 25ul for 384 well plates)
3. Measure the firefly luciferase luminescence on a luminometer. The integration time can be 1 sec to 2 min depending on the luciferase expression level and instrument sensitivity. For most luminometers (Berthold Luminometer, LJL Analyst, Top Count, MicroBeta Counters, CLIPR and LeadSeeker), integration 1 to 5 sec is appropriate
4. Add reconstituted RL Reagent to each well and gently tap to mix. (100ul for 96 well plates, 25ul for 384 well plates)
5. Measure Renilla luciferase luminescence on a luminometer.
General Considerations:Incubation time. Both luciferase reactions are fast, so it is recommended that samples are read as soon as possible after addition of each reagent, preferably within 1 min
Cell number. For assay optimization, it is recommended that the optimal number of cells per well be determined by serial dilution of cells.
Data Analysis:The light intensity (RLU) is directly proportional to the luciferase concentration. For each individual sample, the RLU of the experimental reporter should be normalized by RLU of the control reporter.
