The luciferase-based reporter assay is one of the most commonly used tools in molecular biology to study gene regulation by a protein of interest on a transcription level.

Although the luciferase reporter assay is relatively easy to perform, issues such as high variations between samples and background luminescence, can raise concern about the reliability of your experimental results. Therefore, this article discusses some common issues and troubleshooting solutions when performing the assays.

Troubleshooting: Luciferase Reporter Assays

Below are some common issues when performing luciferase assays:

1. Weak signal

Weak or no signal can occur when your reagents are not functional, your transfection efficiency is low, or the promoter is weak.

To fix this problem:

  • Check if your reagents are still functional and check the quality of your plasmid DNA.
  • Scale up the volume of your sample and reagents per well.
  • Redo the transfection by testing different ratios of plasmid DNA and transfection reagents to find the optimal efficiency, if your transfection efficiency is the problem. The signal from your samples must be above the background signal and your negative control.
  • Replace the weak promoter with a stronger promoter, if possible.

2. High Signal

High signal can happen due to a strong activity of your promoter. One way to overcome this is by diluting your lysate. Perform a serial dilution with your samples to find the dilutions that give optimal results.

3. High Background

If your experiments have high background, reduce the visibility and background issues by using white plates with clear bottoms. If contamination causes the high background, use newly prepared reagents and fresh samples.

4. High Variability

Conditions within each well in a plate can cause variability between replicates or experiments performed at different times. This variability can be the result of pipetting errors, using old reagents, or using a different batch of reagents for different experiments.

Some ways to fix this problem:

  • Prepare a master mix for your working solution.
  • Use a calibrated multichannel pipette.
  • Use a luminometer with an injector to dispense your bioluminescent reagent.
  • Normalize your experiments. For example, by using an internal control reporter in a dual luciferase assay system.

dual reporter luciferase assay, luciferase assay, luciferin, renilla, dual assay

Dual Luciferase Assay System. The dual luciferase assay is an assay using sequential measurements of the firefly and Renilla luciferase activities from the same sample. The results are the ratio of firefly to Renilla luciferase activity, used for normalizing your data and solving the problem with high variability.

5. Interference of the Bioluminescent Signal

Some compounds interfere with the luciferase signal by inhibiting the luciferase enzyme or the substrate. For example, resveratrol or particular flavonoids in the samples inhibit the catalytic activity of luciferase (Braeuning, 2014). Therefore, the use of this compound could affect your experimental findings.

Another example of signal interference: some compounds could quench firefly bioluminescent signal. Particularly, some dyes in blue, black, or red at concentrations greater than 10µM (Auld & Inglese, 2004). Whereas, at high concentrations, some yellow or brown dye compounds could interfere with Renilla luciferase signal (Auld & Inglese, 2004).

Some approaches to reduce the risk of signal interference (Braeuning, 2014).

  • Avoid the use of these inhibitors
  • Use proper controls
  • Modify the time of incubation
  • Lower the concentrations of the compound

6. Stability of Bioluminescent Reagents

Before performing your luciferase assays, you should prepare your reagents ahead of time. However, keep in mind that luciferin and coelenterazine could lose their efficiency over time, before you get a chance to measure the signal.

To avoid this:

  • Check when your bioluminescent reagent starts to lose 50% of the efficiency (or protein half-life) and try to measure the signal before the half-life.
  • Use your newly prepared luciferin or coelenterazine immediately.
  • Use a luminometer with an injector to dispense your reagent.

Tips to Avoid Common Problems during Luciferase Assays

- Test your transfection efficiency and use the best ratio of transfection reagent to DNA.

- Normalize your data by using a secondary reporter.

- Avoid freezing and thawing your samples too many times.

- Avoid using compounds that could inhibit your luciferase assays.

- Use newly prepared luciferin and coelentrazine, cover them with aluminum foil, and store them on ice for immediate use.

Related Articles

To learn more about luciferase assays, check our other articles:

10 Things and Beyond to Consider When Shopping or Using Luciferin/Luciferase

A Crash Course on Luciferase Assays

A Deep Dive Into the Luciferase Assay: What It is, How It Works and More

Why Using a Luciferase Assay Kit Safeguards your Research

Related Product

Browse GoldBio Luciferase Assay Kits:

Illumination™ Renilla Luciferase Enhanced Assay Kit (Catalog ID: I-925)

Illumination™ Firefly & Renilla Luciferase Enhanced Assay Kit (Catalog ID: I-920)

Illumination™ Firefly Luciferase Enhanced Assay Kit (Catalog ID: I-930)

Illumination™ Lyophilized Firefly Luciferase Enhanced Assay Kit (Catalog ID: I-935)


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‌Thorne, N., Inglese, J., & Auld, D. S. (2010). Illuminating Insights into Firefly Luciferase and Other Bioluminescent Reporters Used in Chemical Biology. Chemistry & Biology, 17(6), 646–657.