Simultaneous dual-emission detection of luciferase assays

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Simultaneous dual-emission detection of luciferase reporter assays

Performing dual-spectral luciferase assays using the BMG Labtech POLARstar Omega plate reader.

Megan Dobbs, B.S.; Douglas Hughes, Ph.D.; Janaki Narahari, Ph.D.; Jae Choi, Ph.D.; Georgyi Los, Ph.D.; Brian Webb, Ph.D.; Eric Matthew, M.S.1;

January 24, 2013


Luciferase-based reporter assays are a powerful tool for monitoring gene expression in cells because of their ultrasensitive detection capacity and wide dynamic range. These assays involve inserting a genetic regulatory element upstream of a luciferase gene and then transferring the resulting reporter construct into animal cells, plant cells or bacteria via transfection, transformation or injection. Expression (as luminescence) of the luciferase reporter gene is then measured to quantify the activity of the regulatory element (cis-acting) or proteins (trans-acting) in the biological pathway affected by the target element (Figure 1). Researchers commonly perform dual assays in which the activity of one luciferase reporter is used to measure specific experimental conditions while the activity of a second “control” luciferase measures the transfection efficiency and cell viability.

Figure 1. Schematic of a luciferase reporter assay.

Figure 1. Schematic of a luciferase reporter assay.

The Thermo Scientific Pierce Luciferase Dual Assays deliver high sensitivity in a simple one-step detection protocol. These dual assay kits use pairs of luciferase enzymes that are spectrally resolved (i.e., they luminesce at different wavelengths), allowing their activities to be distinguished and measured in the same samples (Figure 2). This eliminates the usual quenching step needed to differentiate one luciferase signal from the other. The family of Pierce Luciferase Dual Assays includes three dual assay systems in which red firefly luciferase is paired with Cypridina, Gaussia or green Renilla luciferase.

Figure 2. Spectral emission profiles of luciferases used in Thermo Scientific Pierce Dual Assay Kits.

Figure 2. Spectral emission profiles of luciferases used in Thermo Scientific Pierce Dual Assay Kits. Shaded regions indicate the filter sets used in product development: Cypridina Luc and Gaussia Luc (480 +/- 20nm bp), Green Renilla Luc (525 +/- 15nm bp), and Red Firefly Luc (640nm lp).

The BMG LABTECH POLARstar Omega multimodal plate reader is ideal for monitoring activity from two luciferases in a spectrally-resolvable manner (Figure 3). The instrument has a unique simultaneous dual-emission detection mode whereby two photomultiplier tubes (PMT) enable the light from both luciferase reporters to be measured simultaneously with filters. Using this feature of the POLARstar Omega plate reader, we have demonstrated the sensitivity of the Pierce Luciferase Dual Reporter Assays over at least a 100,000-fold luciferase concentration range after normalization to a control reporter, typically red firefly luciferase.

Figure 3. BMG LABTECH POLARstar Omega.

Figure 3. BMG LABTECH POLARstar Omega. The simultaneous dual-emission detection mode of this instrument is ideally suited for use with Pierce Luciferase Dual Reporter Assays. Light emission from both luciferase reporters can be measured simultaneously.


RESULTS and DISCUSSION:

Our experiments confirm the efficacy of Pierce Luciferase Dual Assay Kits to spectrally resolve and simultaneously measure the activity of two luciferases with the POLARstar Omega plate reader. Using the appropriate filters (see figures below), we were able to easily measure the separate luminescence signals in each of the three pairs of luciferase reporter enzymes in the Pierce Dual Assay Kits. The red-shifted light emission of Red Firefly Luc (λmax = 613nm) enables spectral resolution from the blue-emitting Cypridina Luc (λmax = 463nm) and Gaussia Luc (λmax = 470nm), and the green-emitting Green Renilla Luc (λmax = 535nm). This wavelength-differentiation and the stable high activity of Pierce Luciferases enabled sensitive detection over a broad dynamic range. We accurately measured luciferase activities over a 100,000-fold concentration range in the Pierce Luciferase Dual Assays (Figures 4, 5 and 6).

Defining the limit of detection in a dual assay is difficult (Michelini et al). In an ideal two color system, there would be no overlap of emission spectra for one luciferase reporter to the other, allowing for complete wavelength separation with filters. Gaussia and Cypridina luciferase activities have minimal spectral overlap with that of red firefly (Figure 1). By selecting filters for the luciferases in each pair that eliminate or greatly reduce interference between them, conditions for each dual-color assay system can be found that provide accurate measurement over a large dynamic range for each reporter (Figures 4 and 5). When spectral overlap is present between reporters, such as with green Renilla and red firefly luciferases, filter selection is critical for eliminating interference. Alternatively, a corrective calculation may be use (see Pierce Dual-Spectral Luciferase Calculator). For the Renilla-firefly assay, we used a red-shifted 670 +/- 10nm band-pass filter, which is suboptimal for red firefly luciferase (λmax = 613nm) but minimizes interference from green Renilla. Interference was observed only when the light output from green Renilla was significantly greater than the light output from red firefly (Figure 6). The limit of detection and dynamic range of each luciferase is dependent upon luciferase concentration.

A.Figure 4a. Cypridina-Firefly Dual-Spectral Luciferase Assay.

B.Figure 4b. Cypridina-Firefly Dual-Spectral Luciferase Assay.

Figure 4. Cypridina-Firefly Dual-Spectral Luciferase Assay. The Pierce Cypridina-Firefly Luciferase Dual Assay Kit was used to assay activity from each reporter on the POLARstar Omega reader. Using the instrument’s dual-emission optics, light was captured simultaneously through a 475 +/- 30nm band-pass filter (Cypridina Luc) and 610nm long-pass filter (Red Firefly Luc) using a 1000 ms integration time.  A. Serial dilutions (1:10) of HEK293 cell lysate from a stable cell line expressing Cypridina Luc were loaded on a 96-well white plate (10µL/well, n = 6). HEK293 cell lysate from a stable cell line expressing Red Firefly Luc was also added to each well (10µL/well) as a control. B. Serial dilutions (1:10) of HEK293 cell lysate from a stable cell line expressing red firefly luciferase were loaded on a 96-well white plate (10µL/well, n = 6). HEK293 cell lysate from a stable cell line expressing Cypridina Luc was also added to each well (10µL/well) as a mock “control”.

Figure 5. Gaussia-Firefly Dual-Spectral Luciferase Assay.

Figure 5. Gaussia-Firefly Dual-Spectral Luciferase Assay. Serial dilutions (1:10) of HEK293 cell lysate from a stable cell line expressing Gaussia Luc were loaded on a 96-well white plate (10µL/well, n = 6). HEK293 cell lysate from a stable cell line expressing Red Firefly Luc was also added to each well (10µL/well) as a control. The Pierce Gaussia-Firefly Luciferase Dual Assay Kit was used to assay activity from each reporter on the POLARstar Omega reader. Using the instrument’s dual-emission optics, light was captured simultaneously through a 475 +/- 30nm band-pass filter (Gaussia Luc) and 610nm long-pass filter (Red Firefly Luc) using a 1000 ms integration time.

Figure 6. Renilla-Firefly Dual-Spectral Assay.

Figure 6. Renilla-Firefly Dual-Spectral Assay. Serial dilutions (1:10) of HEK293 cell lysate from a stable cell line expressing Green Renilla Luc were added to wells in a 96-well white plate (10µL/well, n = 6). HEK293 cell lysate from a stable cell line expressing red firefly luciferase was also added to each well (10µL/well) as a control. The Pierce Renilla-Firefly Luciferase Dual Assay Kit was used to assay luciferase activities on the POLARstar Omega reader. Using the dual-emission optics, light was captured simultaneously using a 515 +/- 30nm band-pass filter (Green Renilla Luc) and 670 +/- 10nm band-pass filter (Red Firefly Luc) using a 1000ms integration time. The shaded region indicates significant bleed-through of green Renilla luminescence into the red filter. A corrective calculation is needed to separate spectral data in this region (see Calculator).


CONCLUSIONS:

The Thermo Scientific Pierce Luciferase Dual Reporter Assays are sensitive over at least a 100,000-fold dilution range of each luciferase when measured using appropriate filters and the BMG Labtech POLARstar Omega multimodal plate reader.


METHODS:

Materials:

  • HEK293 stable cell lines expressing Cypridina, Gaussia, Green Renilla and Red Firefly luciferase under the control of CMV promoter
  • Pierce Luciferase Cell Lysis Buffer (Part No. 16189)
  • Pierce Cypridina-Firefly Luciferase Dual Assay Kit (Part No. 16184)
  • Pierce Gaussia-Firefly Luciferase Dual Assay Kit (Part No. 16182)
  • Pierce Renilla-Firefly Luciferase Dual Assay Kit (Part No. 16186)
  • White, 96-well f-bottom plates (Thermo Scientific, Cat # 7571)
  • BMG LABTECH POLARstar OMEGA plate reader

HEK293 stable cell lines expressing Cypridina, Gaussia, green Renilla and red firefly luciferases under the control of CMV promoter were lysed with Pierce Luciferase Cell Lysis Buffer. Cell lysates were serial diluted 1:10 in lysis buffer. Replicates of each serial dilution (10µL/well) were loaded into white, 96-well plates. As a control, a cell lysate containing a different second reporter (10µL/well) was also added to each well. For serial dilutions of cell lysate containing Cypridina, Gaussia, or green Renilla luciferase, the second control reporter was red firefly. For serial dilutions of cell lysate containing red firefly, the second control reporter was Cypridina luciferase. Plates were loaded into the BMG LABTECH POLARstar Omega plate reader. The appropriate Pierce Luciferase Dual Assay Working Solution was prepared for each assay, and the instrument injector was primed with working solution. Working solution (50µL/well) was injected into each well. Immediately after reagent addition, luminescence was read through appropriate filters using the plate reader’s simultaneous dual emission mode with a 1 second integration time.


GENERAL REFERENCES:

  1. Michelini, E., et al. (2008). Spectral-resolved gene technology for multiplexed bioluminescence and high-content screening. Anal Chem 80:260-7.

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