Implementation of Promega‘s Dual­Luciferase Reporter Assay System on Tecan’s Spark 20M Multimode Reader

Genetic reporter systems have considerably influenced the study and understanding of cellular responses, gene regulation, and gene expression in both prokaryotic and eukaryotic cells.

A genetic reporter system is made up of a genetic element called a promoter, which is linked to a reporter gene in an expression vector. It is possible to evaluate the reporter protein’s expression by determining the protein’s enzymatic activity or the protein itself.

Enzymatic assays are known to be extremely sensitive, because the reaction product can be produced using just a minimal amount of the reporter enzyme. Luciferase enzymes present in a wide range of organisms have gained popularity in reporter systems, because of their inbuilt sensitivity and effortless measurement.

Assay principle

Dual-Luciferase Reporter Assay System from Promega has been designed to measure a couple of luciferase activities. One reporter enables measuring the response of the experimental target, generally referred to as the ‘experimental reporter’, while the other reporter acts as an internal control to standardize the data received from the experimental reporter.

The firefly beetle (Photinus pyralis) contains Firefly luciferase - a 61 kDa monomeric enzyme that is capable of catalyzing the oxidation of luciferin while discharging light at about 560 nm.

The smaller Renilla luciferase (31 kDa), also a monomeric enzyme from the sea pansy (Renilla reniformis), oxidizes coelenterazine and gives out light centered at 480 nm, as shown in Figure 1. Based on the experimental set-up, the Renilla luciferase or firefly luciferase can be used as the control or experimental reporter.

Firefly and Renilla luciferase reactions.

Figure 1. Firefly and Renilla luciferase reactions.

With regard to the Dual-Luciferase Reporter Assay System, the working of Renilla luciferase and firefly luciferase were sequentially determined. Wells containing a cell lysate sample were injected with 100 μl of the firefly luciferase reagent (LAR II).

The light output over 10 seconds was determined, followed by injecting 100 μl of Stop & Glo® reagent. This halts or quenches the first reaction, and provides the required substrate for the Renilla luciferase reaction.

Measurement was repeated for the light output over 10 seconds. This article explains the implementation of the DLR Assay System on Tecan’s high-end multimode reader, the Spark 20M. It can easily execute high sensitivity flash and glow luminescence measurements with a vibrant range of over nine orders of magnitude.

In addition, its improved luminescence module is capable of measuring multicolor assays with up to five varied luciferases, and also provides spectral scanning of luminescent substances.

Materials and methods

The following materials are used in this analysis:

  • Spark 20M multimode reader (Tecan, Austria)
  • Dual-Luciferase Reporter Assay System (Promega, Germany)
  • 96-well, flat bottom, white polystyrol microplates (Greiner Bio-One, Germany)
  • QuantiLum® Recombinant Luciferase (Promega, Germany))
  • R. reniformis luciferase, recombinant (LUX Biotechnology, UK)

Reagent preparation

Passive lysis buffer (1 x PLB) and LAR II, Dual-Glo® Stop & Glo reagent were prepared by following the manufacturer’s instructions. The next step was preparing stock solutions of firefly and Renilla luciferases (~1 mg/ml protein concentration) with 1 x PLB, followed by diluting each luciferase (around eight million counts) from the stock solutions with 1 x PLB; if needed, further dilution was carried out in 1 x PLB.

Injector A and injector B were then primed with LAR II and Stop & Glo reagent, respectively. The plate was kept within the reader for 10 minutes before conducting the subsequent assay so that the auto­luminescence from the microplate can be reduced.

Tubing adsorption

The adsorption of the reagents onto the inner surface of the injector tube was prevented by pipetting 20 μl aliquots of a 1:50 dilution of recombinant firefly luciferase and Renilla luciferase into one row (12 wells) of a 96-well microplate. After placing the microplate into the reader, the measurement was initiated using the instrument settings listed in Table 1 (Read 1).

After a 10-minute incubation period, the measurement (Read 2) was performed once again using the same parameters after pipetting each dilution into another row (12 wells) of the plate. Equation 1 was then used to calculate the activity of the luciferases in the two measurements.

Activity (%) = Read 2/Read 1 * 100           Equation 1

Where, Activity (%) is luciferase activity, Read 1 is first read and Read 2 is second read after a 10-minute incubation period.

Table 1. Instrument settings for DLR measurements

Parameter

Instrument settings

Plate

Greiner96fw

Mode

Luminescence (well-wise)

Inject

Injector A; 100 μl LARII; 200 μl/s; refill after every injection

Wait

3 seconds

Measure

Luminescence

Attenuation

Automatic

Integration time

10,000 ms

Settle time

0 ms

Output

Counts/second

Inject

Injector B; 100 μl Stop & Glo; 200 μl/s; refill after every injection

Wait

3 seconds

Measure

Luminescence

Attenuation

Automatic

Integration time

10,000 ms

Settle time

0 ms

Output

Counts/second

Quenching

Each well of row A of the microplate was then filled in with 20 μl of PLB as a blank, followed by pipetting 20 μl aliquots of recombinant firefly luciferase into rows D and G (24 wells each). The quenching rate was then determined by measuring the plate using the instrument settings presented in Table 1. Equation 2 was used to calculate the quenching rate.

Quenching = signal FF/signal REN           Equation 2

Where, signal FF is the luminescence measured after injection of LAR II and signal REN is the luminescence measured after injection of Stop & Glo.

Consistency

Dilution of recombinant firefly luciferase with Renilla luciferase was carried out to reach a molar ratio of 1:50 or 50:1. For each of the two conditions, the two rows (24 wells) of a microplate were filled in with 20 μl aliquots of the respective enzyme dilutions. For each luciferase, the consistency of the luminescent signal was then determined by measuring the plate using the parameters listed in Table 1. Equation 3 was used to calculate the consistency.

% CV = st dev/average * 100               Equation 3

Where, % CV is the relative coefficient of variation, stdev is the standard deviation of the signal, and average is the average signal (counts/second).

Measurement parameters

Using the parameters summarized in Table 1, the sequential luminescence measurements were then carried out.

Results

Tubing adsorption

The DLR reagents in the injector tubes were then subjected to a 10-minute incubation in order to calculate the activity of firefly and Renilla luciferase. An activity of ≥95% was observed for both luciferases after subjecting them to a 10-minute incubation period, indicating the absence of adsorption of the reagent to the tubes (Table 2).

Table 2. Tubing adsorption results

FF values

REN values

Average

St dev

Average

St dev

Read 1

8,019,693

227,294

194,715

5,704

Read 2

(after 10 minutes)

7,878,739

50,788

196,234

1,535

Activity (%)

98.24

100.78

Quenching

After the Stop & Glo reagent was injected, it was necessary to quench the firefly luciferase signal ≥10,000 fold (1x104), thereby enabling the detection of only the luminescence of the Renilla luciferase. This experiment involved quenching the signal by an average of ~100,000 fold (1.4x105) for all of the wells analyzed (Figure 2).

Example data from a quenching experiment. The firefly luciferase reaction was quenched after addition of 100 μl Stop & Glo reagent.

Figure 2. Example data from a quenching experiment. The firefly luciferase reaction was quenched after addition of 100 μl Stop & Glo reagent.

Consistency

For the two luciferase reactions, the measurement consistency was demonstrated by testing different enzyme concentrations. Figure 3 depicts the light outputs of the firefly and Renilla luciferase when diluted to different molar ratios (1:50 and 50:1 respectively) and sequentially measured.

No interference was observed between the light outputs of the two different enzymes. Moreover, consistency was observed between the signals measured over 24 wells, with relative coefficients of variation (% CV) in the range of 1.3% - 2.1%.

Firefly and Renilla luciferase signal independence. Firefly and Renilla luciferase were mixed to achieve 1:50 and 50:1 molar ratios, respectively and luminescence was measured in counts/s.

Figure 3. Firefly and Renilla luciferase signal independence. Firefly and Renilla luciferase were mixed to achieve 1:50 and 50:1 molar ratios, respectively and luminescence was measured in counts/s.

Conclusion

This article has demonstrated how Promega‘s Dual-Luciferase Reporter Assay System has been implemented on the Spark 20M multimode reader. The Spark 20M has all the features required to carry out the assay, including high measurement uniformity and an injector module.

Since the luciferase reaction is temperature dependent like many other enzymatic reactions, it is recommended to allow the instrument to reach equilibration with the assay reaction temperature for at least 30 minutes.

A homogenous temperature is maintained within the instrument across the assay, thanks to Spark 20M’s temperature control. A unique cooling module is also offered to offset increases in ambient temperature while performing the measurement.

Proper injector maintenance is important owing to the high sensitivity of luciferase assays. It is necessary to carefully clean the injectors before running DLR assays, so that reagent contamination or dilution can be prevented. Using the ‘backflush’ option, all the liquid can be removed from the tubing system before the injectors are primed with the assay reagents.

Once the assays are performed, it is important to clean the injectors and tubing with distilled water, then with 70% ethanol for 30 minutes, and finally with distilled water. As a result, the Stop & Glo reagent can be efficiently removed as this reagent can be reversibly adsorbed by certain plastic materials.

Depending on the material, some microplates may show significant background auto­luminescence. Therefore, it is necessary to evaluate such microplates before proceeding with an analysis. Some equilibration time must be given to all white assay plates inside the reader in order to decay this background luminescence.

Acknowledgements

Tecan would like to thank Tracy Worzella and Amy Landreman from Promega Corporation for their valuable input and support in this work.

References

  • Spark 20M Brochure. Tecan 398983 V1.0 01-2016
  • Dual-Luciferase Reporter Assay System, Instructions for use (Promega Corp., MA)

Tecan

About Tecan

Tecan is a leading global provider of automated laboratory instruments and solutions. Their systems and components help people working in clinical diagnostics, basic and translational research and drug discovery bring their science to life.

In particular, they develop, produce, market and support automated workflow solutions that empower laboratories to achieve more. Their Cavro branded instrument components are chosen by leading instrumentation suppliers across multiple disciplines.

They work side by side with a range of clients, including diagnostic laboratories, pharmaceutical and biotechnology companies and university research centers. Their expertise extends to developing and manufacturing OEM instruments and components, marketed by their partner companies. Whatever the project – large or small, simple or complex – helping their clients to achieve their goals comes first.

They hold a leading position in all the sectors they work in and have changed the way things are done in research and development labs around the world. In diagnostics, for instance, they have raised the bar when it comes to the reproducibility and throughput of testing.

In under four decades Tecan has grown from a Swiss family business to a brand that is well established on the global stage of life sciences. From pioneering days on a farm to the leading role our business assumes today – empowering research, diagnostics and many applied markets around the world


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Last updated: Apr 1, 2019 at 5:10 AM

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