Shining Stars: Cool NanoLuc® Plasmid Constructs Available Through the Addgene Repository

Researchers having been sharing plasmids ever since there were plasmids to share. Back when I was in the lab, if you read a paper and saw an interesting construct you wished to use, you could either make it yourself or you could “clone by phone”.  One of my professors was excellent at phone cloning with labs around the world and had specific strategies and tactics for getting the plasmids he wanted. Addgene makes this so much easier to share your constructs from lab to lab. Promega supports the Addgene mission statement: Accelerate research and discovery by improving access to useful research materials and information.  Many of our technology platforms like HaloTag® Fusion Protein, codon-optimized Firefly luciferase genes (e.g., luc2), and NanoLuc® Luciferase are present in the repository. We encourage people to go to Addgene to get new innovative tools. Afterall, isn’t science better when we share?

I’d like to focus on some tools in the Addgene collection based on NanoLuc® Luciferase (NLuc).  The creation of NanoLuc® Luciferase and the optimal substrate furimazine is a good story (1).  From a deep sea shrimp to a compact powerhouse of bioluminescence, NLuc is 100-fold brighter than our more common luciferases like firefly (FLuc) and Renilla (RLuc) luciferase.  This is important not so much for how bright you can make a reaction but for how sensitive you can make a reaction.  NLuc requires 100-fold less protein to produce the same amount of light from a Fluc or RLuc reaction.  NLuc lets you work at physiological concentrations.  NLuc is bright enough to detect endogenous tagged genes generated through the CRISPR/Cas9 knock-in.  NLuc is very inviting for endogenous tagging as it is only 19kDa.  An example is the CRISPaint-NLuc construct (Plasmid #67178) for use in the system outlined in Schmid-Burgk, J.L. et al (2).

Two applications of NanoLuc® Technology have caught my attention through coupling the luciferase with fluorescent proteins to make better imaging reporters and biosensors. Continue reading

The Role of the NanoLuc® Reporter in Investigating Ligand-Receptor Interactions

Luminescent reporter assays are powerful research tools for a variety of applications. Last March we presented a webinar on this topic, Understanding Luminescent Reporter Assay Design, which proved to enlighten many who registered. The webinar addressed the importance of careful experimental design when using a luminescent reporter such as Promega’s Firefly or NanoLuc® Luciferase.

Reporters provide a highly sensitive, quantifiable metric for cellular events such as gene expression, protein function and signal transduction. Luminescent reporters have become even more valuable for live, real-time measurement of various processes in living cells. This is backed by the fact that a growing number of scientific publications reference the use of the NanoLuc® Luciferase reporter and demonstrate its effectiveness as a reporter assay. Continue reading

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

6 + 1 Ways Dual-Reporter Assays Can Save Your Data

Dual-Reporter-AssayTransient transfection is often used to perform reporter assays.  We have advocated using a dual-reporter system for decades to normalize the data obtained and gain a clearer understanding of your results.  The experimental reporter should vary with treatment and the control reporter should vary little with treatment. The control reporter thus serves as a marker to help you understand the relative activity of your experimental reporter. Here are seven ways in which dual-reporter assays help you avoid misinterpreting results.

Simply comparing the ratio of the experimental to the control reporter can resolve differences in:

  1. Number of Cells/Well: When manually pipeting cells into a 96-well plate, there is always a chance of having variable numbers of cells in each well. This variation is cell number will affect the experimental and control reporters equally, so the ratio of experimental:control reporter activity will eliminate false interpretation of the experimental data–whether it affects an entire row or column on the plate or individual wells.
  2. Transfection Efficiency: The variations in transfection efficiency will equally affect both the experimental and control reporters so the ratio will normalize the data.
  3. Continue reading

Recommendations for Normalizing Reporter Assays

Reporter assays can be used to investigate a variety of questions from cell signaling to transcription. Your controls depend upon the question you are trying to answer.

As a technical services scientist, I get to hear about many amazing experiments at the planning stage, and I often talk to researchers about how to plan a reporter assay. For the uninitiated, reporter assays are used to “report” the ability or the efficacy of the inserted DNA element to induce/ regulate gene expression as a qualitative or quantitative measure. A typical experimental protocol involves cloning of a DNA fragment upstream of a reporter gene in a plasmid, (and of course confirming the clone by sequencing), transfecting a mammalian cell line with the plasmid and assaying for reporter gene expression by measuring fluorescence, luminescence or absorbance signals. A positive signal would indicate that the cloned DNA element is responsible for driving the gene expression of the reporter.

As in any biological experiment, the controls are as important, if not more, than the actual samples. There are multiple options, and researcher needs to choose the controls depending on the question they would like to ask. Continue reading