Tips for Successful Dual-Reporter Assays

Updated 02/12/2021

Previously, we described some of the advantages of using dual-reporter assays (such as the Dual-Luciferase®, Dual-Glo® Luciferase and the Nano-Glo® Dual-Luciferase® Systems). Another post describes how to choose the best dual-reporter assay for your experiments. For an overview of luciferase-based reporter gene assays, see this short video:

These assays are relatively easy to understand in principle. Use a primary and secondary reporter vector transiently transfected into your favorite mammalian cell line. The primary reporter is commonly used as a marker for a gene, promoter, or response element of interest. The secondary reporter drives a steady level of expression of a different marker. We can use that second marker to normalize the changes in expression of the primary under the assumption that the secondary marker is unaffected by what is being experimentally manipulated.

While there are many advantages to dual-reporter assays, they require careful planning to avoid common pitfalls. Here’s what you can do to avoid repeating some of the common mistakes we see with new users:

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I Have My Luciferase Vector, Now What?

Choosing and Optimizing Transfection Methods

Here in Technical Services we often talk with researchers at the beginning of their project about how to carefully design and get started with their experiments. It is exciting when you have selected the Luciferase Reporter Vector(s) that will best suit your needs; you are going to make luminescent cells! But, how do you pick the best way to get the vector into your cells to express the reporter? What transfection reagent/method will work best for your cell type and experiment? Do you want to do transient (short-term) transfections, or are you going to establish a stable cell line?

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Why You Don’t Need to Select a Wavelength for a Luciferase Assay

It’s a question I’m asked probably once a week. “What wavelength do I select on my luminometer when performing a luciferase assay?” The question is a good and not altogether unexpected one, especially for those new to bioluminescent assays. The answer is that in most cases, you don’t and in fact shouldn’t select a wavelength (the exception to this rule is if you’re measuring light emitted in two simultaneous luciferase reactions). To understand why requires a bit of an explanation of absorbance, fluorescence, and luminescence assays, and the differences among them.

Absorbance, fluorescence, and luminescence assays are all means to quantify something of interest, be that a genetic reporter, cell viability, cytotoxicity, apoptosis, or other markers. In principle, they are all similar. For example, a genetic reporter assay is an indicator of gene expression. The promoter of a gene of interest can be cloned upstream of a reporter such as β-galactosidase, GFP, or firefly luciferase. The amount of each of these reporters that is transcribed into mRNA and translated into protein by the cell is indicative of the endogenous expression of the gene of interest.

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Eight Considerations for Getting the Best Data from Your Luminescent Assays

The stage is set. You’ve spent days setting up this experiment. Your bench is spotless. All the materials you need to finally collect data are laid neatly before you. You fetch your cells from the incubator, add your detection reagents, and carefully slide the assay plate into the luminometer. It whirs and buzzes, and data begin to appear on the computer screen. But wait!

Bad data
These data are garbage!

Don’t let this dramatic person be you. Here are 8 tips from us on things to watch out for before you start your next luminescent assay. Make sure you’ll be getting good data before wasting precious sample!

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Dual-Luciferase or Dual-Glo Luciferase Assay System? Which one should I choose for my reporter assays?

Confused woman

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.

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Executing a NanoBRET™ Experiment: From Start to Data

This is a guest post from Katarzyna Dubiel, marketing intern in Cellular Analysis and Proteomics.

“The objective of my experiment was to test the NanoBRET™ assay as if I was a customer, independent of the research and development team which develops the assay.”

Designing and implementing a new assay can be a challenging process with many unexpected troubleshooting steps. We wanted to know what major snags a scientist new to the NanoBRET™ Assay would encounter. To determine this, we reached out to Laurence Delauriere, a senior applications scientist at Promega-France, who had never previously performed a NanoBRET™ assay. Laurence went step-by-step through the experimental process looking at the CRAF-BRAF interaction in multiple cell lines. In an interview, Laurence provided us with some tips and insights from her work implementing the new NanoBRET™ assay.

In a few words, can you explain NanoBRET?
“NanoBRET is used to monitor protein: protein interactions in live cells. It is a bioluminescence resonance energy transfer (BRET) based assay that uses NanoLuc® luciferase as the BRET energy donor and HaloTag® protein labeled with the HaloTag® NanoBRET™ 618 fluorescent ligand as the energy acceptor to measure the interaction of two binding partners.” Continue reading “Executing a NanoBRET™ Experiment: From Start to Data”

Will This Kit Work with My Sample Type?

Whether you are working with cells, tissues or blood—making sure you use the correct assay system is critical for success.

In Technical Services, we frequently answer questions about whether a kit will work with a particular type of sample. An easy way to find out if other researchers have already tested your sample type of interest is to search a citation database such as Pub Med for the name of the kit and your specific sample type. We also have a searchable peer-reviewed citations database on our web site for papers that specifically cite use of our products. And on many of our product pages, you can find a list of papers that cite use of those products. In Technical Services, we are happy to help you in this search and let you know if scientists here at Promega have tested a particular application or sample type. This information provides a good starting point to optimize your own experiments.

One common question is “can the Caspase-Glo® Assays be used with tissue homogenates?” While Promega has not tested the Caspase-Glo® Assays with tissue homogenates, scientists outside of Promega have used the assays with tissue homogenates with success. As with almost all of our kits, Resources are provided on the catalog page including a list of Citations. As an example, here is a link to the Citations for the Caspase-Glo® 3/7 Assay Systems. We also have an article highlighting a citation on detecting caspase activities in mouse liver. A variety of lysis buffers have been used to make tissue homogenates for this application. To avoid nonspecific protein degradation, it is useful to include a protease inhibitor cocktail in the lysis buffer. The use of protease inhibitors doesn’t usually affect our assay chemistries. Additionally, many commercially available protease inhibitor sets can be used that do not contain caspase inhibitors. It is important to consider the specificity of the kit being used and include proper controls to ensure that the luciferase reaction is performing as expected. For more information on citations and example protocols, feel free to contact us here at Technical Services and we can help get you started with your sample type.

Three Factors That Can Hurt Your Assay Results

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Each luminescent assay plate represents precious time, effort and resources. Did you know that there are three things about your detection instrument that can impact how much useful information you get from each plate?  Instruments with poor sensitivity may cause you to miss low-level samples that could be the “hit” you are looking for.  Instruments with a narrow detection range limit the accuracy or reproducibility you needed to repeat your work.  Finally, instruments that let the signal from bright wells spill into adjacent wells allow crosstalk to occur and skew experimental results, costing you time and leading to failed or repeated experiments.

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For Protein Complementation Assays, Design is Everything

Most, if not all, processes within a cell involve protein-protein interactions, and researchers are always looking for better tools to investigate and monitor these interactions. One such tool is the protein complementation assay (PCA). PCAs use  a reporter, like a luciferase or fluorescent protein, separated into two parts (A and B) that form an active reporter (AB) when brought together. Each part of the split reporter is attached to one of a pair of proteins (X and Y) forming X-A and Y-B. If X and Y interact, A and B are brought together to form the active enzyme (AB), creating a luminescent or fluorescent signal that can be measured. The readout from the PCA assay can help identify conditions or factors that drive the interaction together or apart.

A key consideration when splitting a reporter is to find a site that will allow the two parts to reform into an active enzyme, but not be so strongly attracted to each other that they self-associate and cause a signal, even in the absence of interaction between the primary proteins X and Y. This blog will briefly describe how NanoLuc® Luciferase was separated into large and small fragments (LgBiT and SmBiT) that were individually optimized to create the NanoBiT® Assay and show how the design assists in monitoring protein-protein interactions.

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A Normalization Method for Luciferase Reporter Assays of miRNA-Mediated Regulation

Today’s blog is from guest blogger Ken Doyle of Loquent, LLC. Here, Ken reviews a 2014 paper highlighting specific considerations for using reporter assays to study miRNA-mediated gene regulation.

mirnaThe accelerated pace of research into noncoding RNAs has revealed multiple regulatory roles for microRNAs (miRNAs). These diminutive noncoding RNA species—typically 20-24 nucleotides in length—are now known to mediate a broad range of biological functions in plants and animals. In humans, miRNAs have been implicated in various aspects of development, differentiation, and metabolism. They are known to regulate an assortment of genes involved in processes from neuronal development to stem cell division. Dysregulation of miRNA expression is associated with many disease states, including neurodegenerative disorders, cardiovascular disease, and cancer.

Typically, miRNAs act as post-transcriptional repressors of gene expression, either by targeted degradation of messenger RNA (mRNA) or by interfering with mRNA translation. Most miRNAs exert these effects by binding to specific sequences called microRNA response elements (MREs). These sequences are found most often within the 3´-untranslated regions (3´-UTRs) of animal genes, while they may occur within coding sequences in plant genes.

Studies of the regulatory roles played by miRNAs often involve cell-based assays that use a reporter gene system, such as luciferase or green fluorescent protein. In a standard assay, the reporter gene is cloned upstream of the 3´-UTR sequence being studied; this construct is then cotransfected with the miRNA into cells in culture. A study by Campos-Melo et al., published in September 2014, examined this experimental approach for miRNAs from spinal cord tissues, using firefly luciferase as the reporter gene and Renilla luciferase as a transfection control. Continue reading “A Normalization Method for Luciferase Reporter Assays of miRNA-Mediated Regulation”