Choosing a Luciferase Reporter Assay
A reporter assay comparison guide.
Luciferase Reporters and Detection Reagents
When creating a luciferase reporter assay, there are two important elements to consider: the luciferase reporter protein itself and the assay chemistry used to detect reporter activity. The characteristics of these two components together contribute to the overall performance of the assay. By selecting the reporter/detection solution that is optimal for your experimental goals, you can customize your luciferase reporter assay to create the best solution for your research.
Luciferase Reporter Comparison
Promega offers a choice of 3 different luciferase reporters: NanoLuc® Luciferase, Renilla Luciferase and Firefly Luciferase. See table below to compare their key properties. Learn more in this article: Reporter Genes and Their Applications.
| Luciferase Reporter | |||||
|---|---|---|---|---|---|
| Firefly Luciferase | |||||
| NanoLuc® Luciferase | |||||
| Renilla Luciferase |
*Destabilized versions available to more tightly couple to transcriptional response.
**Secreted versions available.
Detection Reagent Considerations
Multiple assay detection reagents are also available for each reporter. Key considerations for selecting the optimal assay reagent include:
- Signal intensity and overall dynamic range needed for the assay.
- Signal stability, or half-life, which will impact your processing workflow.
- Processing steps required. Non-homogenous assays require a separate lysate creation step prior to reagent addition. Homogenous assay reagents are added directly to the cells in culture eliminating sample pre-processing.
- Lytic or live-cell reporter detection
- Single or dual-reporter detection
Luciferase Signal Strength and Stability
Compare Luciferase Assay Characteristics
| Luciferase Detected | Assay Reagent | Ideal For | Signal Half-Life | Sensitivity | Number of Steps | Live Cell Assay? | Injectors Needed? |
|---|---|---|---|---|---|---|---|
| NanoLuc, Firefly | Nano-Glo® Dual-Luciferase® Reporter Assay System (NanoDLR) | Homogenous Fluc/Nluc dual-reporter detection with flexibility in choice of primary reporter. Provides highest sensitivity for both reporters when a stable signal is required and highest sensitivity option when Nluc is used as the primary reporter. Ideal for low to high-throughput processing when 2 primary reporters or internal control is needed. | 2 hours each | +++++ (Nluc) ++ (Fluc) |
2 (lysate optional) |
No | Optional |
| Firefly, Renilla |
Dual-Luciferase® Reporter Assay System (DLR) | Non-homogenous Fluc/Rluc dual-reporter detection. Requires lysate creation and 2 injectors for delivery. Ideal for small sample numbers when maximum Fluc sensitivity and internal control are needed. | 10 minutes, 2 minutes | ++++ (Fluc) ++++ (Rluc) |
3 | No | Yes 2 injectors |
| Firefly, Renilla |
Dual-Glo® Luciferase Assay System | Homogenous Fluc/Rluc dual-reporter detection with reduced sensitivity and stable signal. Ideal for high-throughput processing of Fluc reporter when Rluc is used as the internal control. | 2 hours each | + (Fluc/Rluc) |
2 | No | No |
| NanoLuc | Nano-Glo® Luciferase Assay System | Homogenous Nluc detection with bright, stable signal. Ideal for low to high-throughput processing when maximum sensitivity is required. | 2 hours | +++++ | 1 | No, unless used with secNluc | No |
| NanoLuc | Nano-Glo® Live Cell Assay System | Live cell Nluc detection with brightest signal. Ideal for single timepoint analysis when high sensitivity is needed in a non-lytic assay. | Up to 2 hours | ++++ | 1 | Yes | No |
| NanoLuc | Nano-Glo® Vivazine™ Live Cell Substrate | Live cell Nluc detection with intermediate signal and stability. Ideal for kinetic analysis lasting multiple hours. | Up to 24 hours | +++ | 1 | Yes | No |
| NanoLuc | Nano-Glo® Endurazine™ Live Cell Substrate | Live cell Nluc detection with most stable signal. Ideal for multi-day kinetic analysis. | Up to 72 hours | ++ | 1 | Yes | No |
| Firefly | Luciferase Assay System | Non-homogenous Fluc detection requiring lysate creation and injector delivery. Ideal for small sample numbers when maximum Fluc sensitivity is needed. | 10 minutes | ++++ | 2 | No | Yes |
| Firefly | Bright-Glo® Luciferase Assay System | Homogenous Fluc detection offering the brightest signal and shortest half-life. Ideal for high-throughput processing when high sensitivity is required. | 30 minutes | +++ | 1 | No | No |
| Firefly | ONE-Glo™ Luciferase Assay System | Homogenous Fluc detection with moderate signal and half-life. Ideal for high- or ultrahigh-throughput processing. | 45 minutes | ++ | 1 | No | No |
| Firefly | ONE-Glo™ Ex Luciferase Assay System | Homogenous Fluc detection with moderate signal and half-life and improved storage stability. Ideal for high- or ultrahigh-throughput processing and repeat use. Also used to detect Fluc in NanoDLR allowing consistent use between single and dual assays. | 2 hours | ++ | 1 | No | No |
| Firefly | Steady-Glo® Luciferase Assay System | Homogenous Fluc detection with maximum signal stability and reduced signal. Ideal for high-throughput applications when extended luminescence is required. | 5 hours | + | 1 | No | No |
| Renilla | Renilla Luciferase Assay System | Non-homogenous Rluc detection requiring lysate creation and injector delivery. Ideal for small sample numbers when maximum Rluc sensitivity is needed. | 2 minutes | ++++ | 2 | No | Yes |
| Renilla | Renilla-Glo Luciferase Assay System | Homogenous Rluc detection with increased signal stability. Ideal for high-throughput processing. | 40+ minutes | ++ | 1 | No | No |
| Renilla | ViviRen™ Live Cell Substrate | Live cell Rluc detection with highest signal. Ideal for single time point or short non-lytic analysis when highest sensitivity is required. | 10 minutes | +++ | 1 | Yes | No |
| Renilla | EnduRen™ Live Cell Substrate | Live cell Rluc detection with greatest stability. Ideal for extended kinetic analysis. | Up to 24 hours | + | 1 | Yes | No |
Bioluminescent Reporter Assay Design
Interested in learning more about reporter assay design? Our two-part "Designing a Bioluminescent Reporter Assay" guide will walk you through basic considerations for choosing the optimal experimental reporter, experimental design and data analysis methods.
Part 1: Choosing Your Experimental Reporter
Part 2: Assay Normalization Options
