I’ve got a set of experiments planned that, if all goes well, will provide me with the answer I have been seeking for months. Plus, my supervisor is eagerly awaiting the results because she needs the data for a grant application, so I don’t want to mess it up. However, I am faced with a choice for my firefly and Renilla luciferase reporter assays: Do I use the Dual-Luciferase® Reporter Assay System or Dual-Glo® Luciferase Assay System? What’s the difference? How do I decide which to use? I’m so confused! Help!
Sound familiar? Not to worry! The choice is not difficult once you know how these assays work and how they differ.
How They Work
The Dual-Luciferase® Reporter Assay System (DLR® Assay) and Dual-Glo® Luciferase Assay System accomplish the same thing: The sequential quantification of two bioluminescent reporters, firefly and Renilla luciferases, from a single sample. Firefly luciferase is a 61kDa monomeric reporter protein that catalyzes the oxidation of beetle luciferin in a reaction that requires ATP, Mg2+ and O2 and produces light (Figure 1). Renilla luciferase is a 36kDa monomeric protein and catalyzes a bioluminescent reaction that uses O2 and coelenterazine. Both are common and convenient reporter genes. In my hypothetical experiments, the firefly luciferase is used as the “experimental” reporter to monitor changes in expression of my gene of interest, while Renilla luciferase is used as the control reporter to normalize results for any interfering factors such as differences in transfection efficiency or cell viability. However, I could have easily designed my experiments the other way around, with Renilla luciferase as the experimental reporter.
The DLR® and Dual-Glo® Assays both include a firefly luciferase reagent (Luciferase Assay Reagent II for the DLR® Assay and Dual-Glo® Luciferase Reagent for the Dual-Glo® Assay) and a Renilla luciferase reagent (Stop & Glo® Reagent for the DLR® Assay and Dual-Glo® Stop & Glo® Reagent for the Dual-Glo® Assay). For both systems, the firefly luciferase reagent is added first, and luminescence is measured before the Stop & Glo® Reagent is added to quench the firefly luciferase signal and initiate the Renilla luciferase reaction. Luminescence is measured a second time to quantify Renilla luciferase activity.
How They Differ
There are important differences between these assays that will determine which is best for my situation. The DLR® Assay includes a separate lysis buffer, the Passive Lysis Buffer, and requires that I prepare cell lysates prior to the assay. A portion of each lysate is used in the DLR® Assay, and the remaining lysate can be stored at –20° for up to 1 month or at –70°C for long-term storage. The Dual-Glo® Assay does not require a separate lysis buffer. Cell lysis components are included in the Dual-Glo® Luciferase Reagent. Simply add an equal volume of Dual-Glo® reagent directly to the cell culture, and after at least 10 minutes to allow cell lysis, measure luminescence.
Another difference is signal half-life. The DLR® Assay generates luminescence that rapidly decreases in intensity. The firefly luciferase signal decreases 50% in approximately 12–15 minutes, and the Renilla luciferase signal decreases 50% in less than 3 minutes. These signal kinetics make measurement of firefly and Renilla luciferase activities difficult if large numbers of samples are measured in 96- or 384-well plates. Lysates must be assayed one at a time in a tube format, or a luminometer equipped with one or two reagent injectors must be used to accommodate the “read-inject-read” format of the DLR® Assay.
In contrast, the Dual-Glo® Assay has stabilized luminescent signals, with firefly and Renilla luciferase signal half-lives of approximately 2 hours. Thus, the Dual-Glo® Assay can be used to measure both firefly and Renilla luminescence in multiwell plates using a plate-reading luminometer, CCD camera or similar imaging device without injectors. (Note: Injectors should not be used with the Dual-Glo® Assay to avoid foaming, which can interfere with luminescence measurements.) Plates containing experimental samples can be batch processed: I can add the Dual-Glo® Luciferase Reagent to all wells of the plate, measure firefly luminescence across the entire plate, add the Dual-Glo® Stop & Glo® Reagent to all wells of the plate, then measure Renilla luminescence across the entire plate. Due to the 2-hour half-life, I don’t have to worry about significant signal loss as wells wait to be read.
Finally, the light output will be different. Firefly and Renilla luciferases undergo spontaneous inactivation after generating luminescence. This inactivation causes the “flash”-type kinetics characteristic of the DLR® Assay. To generate a more stable luminescent signal that is amenable to high-throughput measurements, the rate of inactivation and subsequently the rate of catalysis must be slowed. For this reason, the Dual-Glo® Assay results in lower luminescence levels than the DLR® Assay, which was developed for maximal sensitivity. This difference in light output will affect my raw data (i.e., the number of relative light units [RLU]). Luminescence levels measured using the DLR® Assay will be dramatically but proportionately higher (approximately 100-fold) than those using the Dual-Glo® Assay.
The background also will be proportionately higher. However, the final results—the trends, as determined by the increase or decrease in the ratio of luciferase activities—will be the same, so does it really make a difference in the end? Do I really care if my samples yield 100,000RLU or 10,000,000RLU if all of the readings, including the background, are proportionally higher? When I worked as a Promega Technical Service Scientist, I would encounter researchers who were obsessed with high luminometer readings, and my coworkers and I would always joke that if someone wanted higher RLU readings for his raw data, he should just tape a piece of paper with three zeroes next to the display of the luminometer; just make sure that the three zeroes are added to all readings, including the background.
Which Assay Do I Pick?
If you are in the same situation as I am, trying to determine which dual-luciferase assay is right for you, ask yourself the following questions:
How many samples am I processing?
The DLR® Assay requires you to prepare a lysate for each sample, which can be stored for later use. The Dual-Glo® Assay offers the convenience of incorporating the cell lysis step into the first step of the reporter assay and processing samples in a multiwell format but consumes the entire sample.
Do I really need those higher luminometer readings, or can I get by with lower readings if it means I have the convenience of performing my assays in multiwell plates?
The DLR® Assay yields higher luminescence readings, but the shorter signal half-lives makes it less suited to high-throughput measurements than the Dual-Glo® Assay.
What luminometer or other imaging device will I use?
The Dual-Glo® Assay does not require a luminometer with injectors due to the longer signal half-lives (in fact, foaming caused by injectors can interfere with luminescence measurements), while the DLR® Assay requires an imaging device that can measure luminescence within seconds of reagent addition.
Now that you have a better understanding of how these two products differ, I want to mention one more option for consideration in the quest to find your best fit dual-luciferase assay system: our latest addition to the dual-luciferase reporter assay family, the Nano-Glo® Dual-Luciferase® Reporter Assay System (NanoDLR™). Similar to the Dual-Glo® Assay, NanoDLR™ has glow signal kinetics but, unlike both the DLR® and Dual-Glo® Assays, the second reporter is our NanoLuc® luciferase, an ATP-independent luciferase engineered from a deep-sea shrimp. The heightened sensitivity, small size and brightness of the NanoLuc® luciferase, in conjunction with improved firefly luciferase chemistry, provides you with flexible assay options that are highly sensitive, easy to use and result in exceptional data quality.
Figure 2. A comparative look at luminescence signals from HEK293 cells transfected with a 1:1:8 ratio of either TK-Rluc (Renilla):TK-Fluc (firefly):carrier DNA or TK-Nluc (NanoLuc):TK-Fluc:carrier DNA and assayed using NanoDLR™, DLR™ or Dual-Glo® Assay Systems, as indicated.
For more information about how these assays work and which one might be best for you, check out the technical literature:
- Dual-Luciferase® Reporter Assay System Technical Manual TM040
- Dual-Glo® Luciferase Assay System Technical Manual TM058
Nano-Glo® Dual-Luciferase® Reporter Assay Technical Manual TM426
- Dual-Glo™ Luciferase Assay System: Convenient dual reporter measurements in 96- and 384-well plates
- NanoLuc™: A Smaller, Brighter and More Stable Luciferase Reporter