Versatile Luciferases: New Tools for Reporter Assays

Microplate luminometers equipped with reagent dispensers and optical filters provide optimal performance for luciferase assays with Cypridina, Gaussia, Renilla and firefly luciferase reporter genes

by Reija-Riitta Harinen, Ph.D.1; Jorma Lampinen, Ph.D.1; Arto Perälä, Ph.D.1; Janaki Narahari, Ph.D.; Douglas Hughes, Ph.D. - 06/24/13

This application note presents how Thermo Scientific microplate luminometers in conjunction with Thermo Scientific Pierce Luciferase Reporter Gene Assays form a powerful combination for reporter gene studies in high-throughput format. The focus of this application note is on introducing different luciferase reporter assay types and the requirements they place on the luminometer. It also shows how luciferase reporter gene assays provide excellent performance using low assay volumes with normal and shallow well 384-well plates.

Luciferase genes are commonly used as reporter genes in gene regulation studies. Reporter gene assays are used both in basic research for studying transcriptional regulation and cell signaling, and in drug discovery for screening and validating drug targets. The light output from luciferase reactions can be detected with luminometers that are suitable for reporter gene assays.

We have developed a family of luciferase activity assays that use novel luciferase genes from Cypridina and Gaussia combined with the Red Firefly luciferase gene and the green-shifted Renillagene. The assay family provides both flash- and glow-type assays, which differ in the stability of luminescence signals and in sensitivity. Some of the assays utilize luciferases that are secreted into the culture media. This feature allows live monitoring of the reporter activity during cell growth, without a need for cell lysis. In addition, the assay family includes unique dual-spectral luciferase assays for multiplexing purposes. The dual assays include two luciferases: one for measuring the experimental luciferase activity and the other for measuring control activity for normalization. See the luciferase reporter assay portfolio in Figure 1.

Figure 1. Thermo Scientific Pierce Luciferase Reporter Assay reagents and luciferase genes.

Glow-type luciferase assays

Glow-type luciferase reporter gene assays produce a very stable luminescence light signal that lasts for approximately one hour. As the light emission decays slowly, measurement does not have to be performed immediately after the addition of substrate. The stability of the light makes it possible to pipette all the assay reagents manually without need of reagent dispensers installed to the instrument. Glow-type assays are generally less sensitive than flash-type assays.

Flash-type luciferase assays

Flash-type luciferase reporter gene assays are in general more sensitive than glow-type assays. Luminescence signals are transient in flash-type assays, so the signal peak is reached soon after assay reagent addition. Therefore, flash-type assays often require the assay reagent to be added with automatic dispensers installed to the luminometer. Reagent dispensers in the instrument facilitate signal monitoring right from the start of the luminescence reaction and enable precise timing between dispensing and measurement from sample to sample. Pipetting the assay reagent manually increases sample-to-sample signal level variations especially in flash-type assays and miss the capture of true flash signal due to delay in measuring the signal after manual reagent addition.

Dual-reporter luciferase assays

Thermo Scientific Luciferase Dual Assays are highly sensitive flash-type assays for multiplexing. The one-step assays are based on having two luciferases emitting luminescent light at spectrally distinct wavelengths. The signals can easily be separated using a luminometer equipped with high transmission filters optimized for the assays. This kind of dual assay eliminates the need for signal quenching between the measurement of two luciferases. Thus both luciferase signals can be detected simultaneously from a single sample with a single reagent addition. As these dual assays are flash-type assays, the reagent dispensers installed to the luminometer act to ensure optimal performance and sensitivity.

Assay Procedures

See the assay procedures of both single and dual luciferase reporter assays in Figures 2 and 3. The protocol of luminescence measurement is very simple: add luciferase samples into a white microplate. In case of a flash-type assay, add the luciferase assay reagent with an automatic dispenser and immediately read the signal for the suggested integration time.

Figure 3. Dual-reporter assay procedure.

Luminometers

Luminescence light output from luciferase reporter gene assays can be quantitated using, for example, Thermo Scientific Varioskan Flash or Luminoskan Ascent microplate luminometers (Figure 4). Thermo Scientific instruments provide the best possible performance with the Pierce Luciferase Reporter Assays. These luminometers have excellent sensitivity in luminometry, both in glow- and flash-type assays. In addition, the Varioskan Flash luminometer can measure luminescence spectra and also other detection technologies besides luminometry.

Reagent dispensers

Both the Luminoskan Ascent and Varioskan Flash microplate luminometers can be equipped with up to three reagent dispensers (Figure 5). Dispensers are essential in flash-type assays, which require measurement soon after the addition of substrate, and precise timing between dispensing and measurement from sample to sample and from day to day. Dispensers installed to the luminometer make the reaction triggering easy, fast and accurate, not only for flash-type assays, but also for glow-type assays.

Optical filters

In addition, both the Luminoskan Ascent and Varioskan Flash instruments can be equipped with optical filters for measuring dual luciferase assays. The filters have been specifically optimized to separate the two luciferase signals as efficiently as possible for the Pierce Dual Luciferase Assays and, therefore, they ensure excellent performance.

The Pierce Luciferase Reporter Assays and the Varioskan Flash luminometer provide very high performance and enable use of very low assay volumes with normal 384-well and shallow well 384-well plates. All luciferase reporter assays show perfect linear response over a wide concentration range with both normal and shallow well plates. Real assay dynamics reaches eight orders of magnitude with the Gaussia and Cypridina flash assays (Figure 6). The Renilla flash assay also shows a remarkably large dynamic measurement range (data not shown). Flash assay with Red Firefly luciferase shows a somewhat reduced dynamic range, about 5 to 6 orders of magnitude (data not shown).

The Varioskan Flash luminometer can automatically adjust the photomultiplier gain to increase the assay dynamic range to eight orders of magnitude. This unique feature means that the luciferase samples can have any luciferase concentration over a large 100 million-fold concentration range. Thus the samples can always be measured without additional dilution steps.

Figure 7 illustrates the flash-type reaction kinetics of Gaussia, Cypridina, Renilla and Firefly luciferases. All the assays reach the light emission maximum in a few seconds after the reaction has been started. Cypridina and Renilla assays show a fairly stable light emission for several seconds, and the Gaussia assay shows about 3% signal decay per second after reaching maximum. The firefly flash assay represents the fastest flash reaction where the signal decays about 20% per second during the first couple of seconds.

Using automatic dispensers of the luminometer for reagent addition is highly recommended to ensure exact timing in the measurements, especially in Gaussia and Firefly assays. If the luminescence reaction is initiated by manual pipetting, it may lead samples to be measured in different time points of the kinetic reaction, thus increasing sample-to-sample variations.

Assay sensitivity is dependent on the measurement integration time, but its effect is not very strong. About a one second measurement time provides good sensitivity, but a few seconds more may be required for the best possible sensitivity. The Cypridina assay produces the most stable light emission and therefore it is recommended to integrate the signal somewhat longer than with assays with a more unstable signal. The firefly flash reaction shows the most unstable kinetics and therefore the recommended integration time is the shortest. A usable integration time range is a range where changing the integration time has only a minor effect on the assay performance. When using shallow well 384-well plates with small assay volumes, it is recommended to use longer integration times than with normal 384-well plates with higher assay volumes. See Table 1.

Table 1. Recommended signal integration times (in seconds) of different luciferase assays and plate formats.

Even though this application note only presents the results of assays performed in a 384-well plate format, measurements in a 96-well plate format provide excellent performance as well. The performance of the Luminoskan Ascent luminometer with these luciferase reporter assays is also comparable to the Varioskan Flash luminometer results presented here.

• Pierce luciferase assays provide an extremely wide dynamic range for measuring both low and high luciferase expression levels without concentrating or diluting samples.
• The Varioskan Flash multimode microplate reader equipped with a luminometric measurement module detects luciferase activities over a dynamic range of about eight orders of magnitude with the automatic gain adjustment feature of the instrument.
• Using 384-well plates (either normal or shallow well) enables reducing both sample and reagent consumption without sacrificing assay performance. Use of shallow well 384-well plates enables performing the assays in a total volume of about 6µL (1µL sample + 5µL assay reagent).
• Highly sensitive flash-type assays can easily be performed with a microplate luminometer equipped with an automatic dispenser.
• Both inter- and intra-assay variations can easily be minimized using automatic luminometer dispensers, which enable precise timing of the reaction start and signal collection.

Different luciferase reporter gene assays require different features and capabilities of the luminometer. The benefits of using Thermo Scientific luminometers for performing luciferase reporter assays include the following:

• Excellent luminometric performance in glow- and flash-type assays
• Onboard dispensers for reagent addition, which provides precise timing between dispensing and measurement in flash assays, as well as convenience and accuracy for flash and glow assays
• Optimized filters for measurement of dual assays
• Flexible and easy protocol setup and data processing with PC software

Luminometer: Thermo Scientific Varioskan Flash multimode microplate reader equipped with a luminometric measurement module and an onboard dispenser for automatic reagent addition

Luciferase assay reagents: Flash-type Thermo Scientific Pierce Luciferase Reporter Assay Kits for Gaussia (Part No.16158), Cypridina (Part No. 16168), Renilla (Part No. 16164) and Firefly (Part No. 16174) luciferases

Microplates: “Normal” plates were OptiPlate™-384 White Opaque 384-well Microplates (Perkin Elmer, #6007299). “Shallow well” plates were Thermo Scientific Nunc 384-well shallow well standard height white shallow plates (#264706).

Procedure: Luciferase samples from were diluted in the Cell Lysis buffer provided in the kit to create a concentration series of over eight orders of magnitude. Aliquots of 4µL (normal 384-well plate) or 1µL (shallow well 384-well plate) of each dilution were added into the microplate wells. Instrument control software was programmed to add 20µL or 5µL of luciferase assay reagent with a dispenser and to measure the signal kinetically for 15 seconds at a sampling rate of ten readings per second. Each well was dispensed and measured before proceeding to the next well.

Analysis: Luminescence signals were integrated from kinetic curves for different time periods (from 0 to 1 s and from 0 to 15 s). Additionally, individual signals and signal maxima were collected for comparison of different time points. Assay sensitivity and dynamic range were calculated for each data set.

This article was first published as Application Note ANMRLuci-1012, October 2012.