A new approach to examine multiple cellular pathways using multiplex luciferases

We at BMG are really looking forward to SLAS2020! Our team will be back in San Diego and ready to meet you and help you to choose the right microplate reader. We know that improving throughput is important to many of you and getting more useful information is always key. Come find out how we can help you with all of your assay needs including luciferase assays.


Dr Carl Peters Dr Carl Peters (6)

As always BMG will have an outstanding slate of talks and posters to help you find out more about the science that is being achieved using our microplate readers. In this post I want to tell you more about the session we are hosting on Monday January 27th from 12:30 to 1:15 in Room 5A at the San Diego Convention Center.

This session will have a presentation by Koen Venken who has been working as an assistant professor at Baylor College of Medicine in Houston, Texas. The goal of Dr. Venken’s work is to build better multiplex systems in order to better understand complex biological responses and using this understanding to enable implementation of genetic and pharmaceutical treatments of aberrant physiology. The Venken labs pursuits recently produced a Nature Communications article: ‘Examining multiple cellular pathways at once using multiplex hextuple luciferase assaying.’

About this work Dr. Venken had this to say:

"The comprehensive analysis of complex cellular signaling events benefits from multiplex luciferase assaying methods that deliver multiple, highly quantitative measurements during a single experiment. Until recently, luciferase multiplexing was limited to two, and just one is used as a pathways reporter (the second luciferase serves as the normalization control). By leveraging differences in wavelength of light released by different luciferases, and the use of different substrates, we were able to expand multiplexing from two up to six, therefore significantly increasing the number of pathways that can be monitored simultaneously in a single experiment. While we employed multiplex hextuple luciferase assaying to monitor changes in the signaling of five cancer pathways against a normalization control, the flexibility of our approach provides immediate opportunities to explore any five pathways for any biomedical problem at the same time."

To understand the significance of the work achieved in this paper it is probably useful to take a step back and think about luciferase reporters. Most of us biologists have probably used a dual-luciferase assay and even more of you are probably at least familiar with this concept which is used in the commercial DLR® from Promega. Cells are transfected with two plasmids for 2 different luciferase enzymes that have distinct substrate requirements to produce light. The elements regulating expression of one luciferase enzyme is designed as a readout for gene expression, characterization of transcription factors and more, while the second luciferase is constitutively expressed to serve as a control for transfection efficiency and cell health. The substrate specificity of the 2 luciferase enzymes, and the ability to quench one of the enzymes activity, means they can be sequentially probed for their level expression, viewed as the light produced.

In the years since dual luciferase assays were introduced many discoveries about luciferase enzymes have been made and genetic modification of enzymes has led to a broader palette of colored light that can be produced by these enzymes. With these facts in mind, Dr. Venken’s group sought to increase the multiplex capacity of luciferase assays. To achieve this they used four criteria to characterize useful luciferases:


  1. Substrate preference and potential for quenching
  2. Luciferases with the same substrate preference must exhibit emission spectra that can be resolved by emission filters or mathematical computation
  3. Stable light emission
  4. Wide dynamic range


The Venken lab took full advantage of the excellent performance available in their CLARIOstar microplate reader which they purchased about 4.5 years ago. This performance was of particular use when they characterized the emission spectrum for each of 12 luciferases with their respective preferred substrates. The Linear Variable Filter emission monochromator was set up to measure from 350 to 700 nm in 1 nm increments. The bandwidth was usually 10 nm but for lower intensity light-emitting luciferases this was increased to 20 nm. These people really know what they are doing when it comes to using the CLARIOstar!

Fig. 1: BMG LABTECH’s Fluorophore Toolbox provides spectra of various luciferases and fluorophores and additionally offers respective filter recommendations. When using the LVF monochromator, the wavelength and bandwidth can be freely selected in the available range with a resolution of 1nm.

All told they were able to identify 6 luciferases of which could employ 2 different substrates such that two groups of 3 luciferases can be sequentially probed for activity. One of these serves as the control so 5 different responses can be assessed in a single experimental sample!

A further feature of the work in this paper is that all 6 luciferase genes are encoded onto a single plasmid, a process they call solotransfection, thus decreasing variability between biological replicates. The approach they use to achieve this plasmid is adaptable so reporters for any other pathway should be easy to implement.

Their proof of principle data showed the ability to detect direct and collateral effects of a variety of pathway specific treatments in a cancer model. But the applications of this technology extend beyond this. Applications in cellular pathways could include assessing ligand specificity for nuclear receptors as well as numerous applications in complex diseases where it is increasingly clear that pharmaceuticals have off-target effects.

But it appears that six is not enough! In the future they hope incorporate more luciferases! Additional luciferases with the same substrate specificity could be employed if their emission spectra are different enough to be distinguished from those used already. In addition luciferases with distinct substrate specificities could be used if they are also able to be quenched.

So, thank you Dr. Venken for your participation at the upcoming SLAS! We are very excited to hear more about what you have done!

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