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Dual-Luciferase Quantitation in the LMax Microplate Luminometer (MaxLine Application Note #39)

Evelyn McGown, Ph.D. and Michael Su, M.S.
Molecular Devices Corporation, 8/00


INTRODUCTION
Reporter gene assays are used to study eukaryotic gene expression. Dual geneticreporters are commonly used in transient transfections of cultured cells tominimize experimental variability caused by differences in cell number, viabilityor transfection efficiency. One vector containing the experimental reporter gene iscotransfected with a second vector containing a different reporter gene to serve asthe control. Typically, the experimental reporter is coupled to a regulatedpromoter, and the control reporter gene is coupled to a constitutive promoter. Bynormalizing the changes in expression of reporter activity to the activity of thecontrol, experimental variability can be minimized.

Colorimetric reporter assays have been widely used, but bioluminescent reportersystems, especially using firefly luciferase, are becoming increasingly popularbecause of their speed, sensitivity and wide dynamic range. PromegaCorporation has recently introduced their Dual-Luciferase Reporter Assay System which utilizes the firefly (Photinus pyralis) and Renilla (Renilla reniformis)luciferases1. Because the enzymes have different substrate requirements, they canboth be measured in a single reaction mixture. The firefly luciferase is typicallyused as the experimental reporter, and the renilla luciferase serves as the control.

Figure 1 illustrates the two enzymatic reactions. Firefly luciferase enzymecatalyzes the oxidation of luciferin with the concomitant release of light2. The reaction requires ATP, Mg+2 and O2. Renilla luciferase catalyzes the O2-dependentoxidation of coelenterate luciferin (coelenterazine) but does not require Mg orATP3. Both reactions can easily be measured in the Molecular Devices Lmaxmicroplate luminometer. An example of results is presented in this application note.


MATERIALS
1. Dual-Luciferase Reporter Assay System kit, Cat #E1960; Promega Corporation. The contents include: Luciferase assay buffer II (E1955); Luciferase assay substrate (E151A), Stop & Glo buffer (E197A); Stop & Glo substrate (E193B); Stop & Glo Substrate Solvent (E463A); Passive lysis buffer, 5x reagent (E194A).

2. Firefly Luciferase, Sigma Cat # 9506.

3. Recombinant Renilla luciferase; Novalite, Cat #4400; Chemicon International; 800-437-7500.

4. Solid white microplates; e.g. CorningCostar Cat. No. 3912, Tel: 1800492 1110.

5. Lmax microplate luminometer with SOFTmax PRO for Lmax (Molecular Devices Corp.)


PREPARATION OF LUCIFERASE STANDARDS
1. Passive lysis buffer was prepared by mixing 2 mL of passive lysis 5x reagent (E194A) with 8 mL deionized water.

2. The firefly luciferase stock (1 mg/mL) was prepared in phosphate-buffered saline (PBS) containing 1% bovine serum albumin (BSA) and frozen in aliquots at -70C until use. When needed, a vial was thawed and a 10 g/mL standard prepared by diluting 10 L of the stock with 990 L passive lysis buffer.

3. The Renilla luciferase stock (10 g/mL) was prepared by adding 990 L passive lysis buffer to a 10 g vial of the enzyme and mixing gently. The solution (10 g/mL) was divided into aliquots and frozen at -70C until use.

4. The dual luciferase working standard #1 was prepared by diluting 10 L each of the 10 g/mL firefly and 10 g/mL Renilla luciferase with 980 L passive lysis buffer (concentration of each enzyme = 100 ng/mL). Serial one-to-ten dilutions in passive lysis buffer achieved working standard concentrations from 105 to 10-1 pg/mL for both enzymes. The diluted standards were kept on ice until use.


PREPARATION OF REAGENTS
1. Luciferase assay reagent II (LAR II) was prepared by resuspending the lyophilized luciferase assay substrate (E151A) in 10 mL luciferase assay buffer II (E1955).

2. The Stop & Glo reagent was prepared by first reconstituting the dried Stop & Glo substrate with 200 L Stop & Glo Substrate Solvent (E463A). After gently vortexing for 10 sec, the entire reconstituted substrate was mixed with 10 mL Stop & Glo buffer (E197A).


PREPARATION OF THE LMAX
1. The solvent delivery systems of the Lmax were both cleaned by pumping 75% ethanol through them and allowing it to stand for one hour, followed by rinsing generously with deionized water (5 washes, 99 cycles each).

2. The P injector was filled with the LAR II reagent by pumping 2 mL through it. The M injector was similarly primed with Stop & Glo Reagent.


ASSAY PROCEDURE
The kit instructions were followed, except that the measurements were made on diluted recombinant enzymes instead of lysed cells.

1. In SOFTmax PRO, Dual Read mode was selected. Both the P and M injectors were set to deliver 100 L/well, followed by a 2-second delay and a 5-second read.

2. The diluted firefly+Renilla luciferase standards and buffer blank were pipetted (20 L each) into microplate wells in quadruplicate. The final enzyme content for each ranged from 2.0 to 2 x 106 fg/well.

3. The plate containing the luciferase standards was placed into the drawer of the Lmax and the injection/read cycles initiated.

During the assay, the P injector supplies the LAR II reagent to each well a minimum of 1.6 sec ahead of the time the read head moves into position over that well. The M injector supplies Stop & Glo directly under the read head.


EXAMPLE OF DUAL LUCIFERASE RESULTS OBTAINED ON THE LMAX
A SOFTmax PRO plate section containing raw dual luciferase data is shown in Figure 2. The instrument settings are displayed to the right, along with the time/ data stamp. In each well, there are 3 numbers: the top is the firefly luciferase value (P injector), the middle is the Renilla luciferase value (M injector), and the bottom number is the ratio of the two. (The ratio was selected in the Reduction dialog box in SOFTmax PRO, and the Display was set to show Raw + Reduced data.)


The two standard curves are shown in Figure 3. The signal from the renilla luciferase was approximately 10 times brighter than that of the firefly enzyme. The calculated limits of detection (amount producing a signal higher than 3 positive SD of the blank values above zero) were approximately 2 and 0.2 fg/well for the firefly and renilla enzymes respectively.


DUAL READ VALUES IN THE GROUP TABLE
Although the examples presented here are standard curves of firefly and renilla luciferase, the purpose of the dual assay is to ascertain the ratios of the two activities. The ratio can be calculated automatically in SOFTmax PRO if you specify the ratio as the desired calculation in the Reduction Dialog Box. If you also wish to display the individual values in the Group Table, this can be easily accomplished as described below.

When you create a group and assign sample locations in Template Editor, SOFTmax PRO automatically creates a group table to list the sample names and the corresponding data. The actual values which appear in the group table depend on which data calculation you have selected in the Reduction dialog box. In the example shown in Figure 2 above, the reduction is set to the ratio of P value/M value, so the ratios appear automatically in the group table. If, instead, the reduction is set to P value or M value, that set of values would appear in the group table.

If you wish to list both the firefly and renilla values in the group table, as well as the ratios, you can create additional columns and type in the desired formulas. Those formulas are summarized in the table below.


An example of a group table containing the 3 values (and other information) is shown in Figure 4. In this example the purified enzyme preparations were diluted in tandem, so the ratios are predictably the same for all of the samples (except the very lowest concentrations). In real cell-based experiments, the ratios would differ.


SUMMARY
The Lmax microplate luminometer is a sensitive and easy-to-use instrument for measuring firefly and renilla luciferase activity in a microplate format. The dual sequential measurements can be automated, and SOFTmax PRO for Lmax offers automatic calculations of the ratios of the two activities as well as additional powerful data analysis capability.


REFERENCES

1. www.promega.com

2. DeLuca, M.A. and W.D. McElroy, (1978) in Meth. Enzymol. vol 53:, p.3

3. Matthews, J.C. et al. (1977) Purification and properties of Renilla reniformis luciferase. Biochemistry, 16:85.


By default, SOFTmax PRO brings only the single set of reduced data values into the group table. If you want additional data listed in the group table (e.g. more than one reading or additional secondary calculations), you must create new columns and specify the desired values by typing in custom formulas. For details, see your SOFTmax PRO Users Manual.


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