Posters
« Back
A New Dual Luciferase Assay Using NanoLuc® Enables a Second Generation Coincidence Reporter System to Reduce False Hits in HTS Poster
EP23509
Poster Title: A New Dual Luciferase Assay Using NanoLuc® Enables a Second Generation Coincidence Reporter System to Reduce False Hits in HTS Poster
Submitted on 09 Oct 2015
Author(s): Christopher Eggers, Samuel Hasson, Brock Binkowski, Matt Robers, James Unch, Braeden Butler, , Keith Wood, James Inglese and Frank Fan
Affiliations: Promega Corporation
Poster Views: 3,377
View poster »


Poster Information
Abstract: Luciferase-based reporter-gene assays remain a cornerstone of high-throughput screening of compounds because of their high sensitivity and dynamic range. However, a substantial number of non-relevant hits can be generated due to direct interaction of compounds with the luciferase reporter. To help differentiate compounds modulating the biological pathway of interest from those affecting the stability or activity of the reporter enzyme, we have developed a second-generation coincidence reporter system in which transcriptional activation leads to stoichiometric expression of two orthologous reporters that have dissimilar profiles of compound interference. In this system, firefly luciferase (Fluc) and PEST-destabilized NanoLuc® luciferase (NlucP) are expressed off the same promoter using ribosome skipping mediated by the P2A peptide.

To sensitively measure both Fluc and NanoLuc (Nluc) in the same sample, we have developed the Nano-Glo® Dual-Luciferase® Reporter (NanoDLR™) Assay System, a homogeneous lytic assay performed in an “add-read-add-read” format, in which the Fluc signal is quenched over a million-fold by addition of the Nluc reagent. The increased brightness of Nluc and improved Fluc inhibition means that Nluc can be detected at over 2-3 orders of magnitude lower molar concentration than Renilla luciferase in the existing homogenous firefly/Renilla dual-luciferase assay (Dual-Glo), allowing both luciferases to be dynamic reporters. Following single-copy integration of the Fluc-2A-NlucP biocircuit into a gene locus relevant to Parkinson’s disease, HTS using NanoDLR easily distinguished compounds affecting one of the reporters from those affecting transcription, yielding a >5-fold decrease in the number of hits.
Summary: Luciferase-based reporter-gene assays remain a cornerstone of high-throughput screening of compounds because of their high sensitivity and dynamic range. However, a substantial number of non-relevant hits can be generated due to direct interaction of compounds with the luciferase reporter. References: Christopher Eggers1, Samuel Hasson2, Brock Binkowski1, Matt Robers1, James Unch3, Braeden Butler1, , Keith Wood1, James Inglese2 and Frank Fan1
1Promega Corporation, 2800 Woods Hollow Rd, Madison, WI 53711-5399; 2National Center for Advancing Translational Sciences, 9800 Medical Center Dr, Rockville, MD 20850; 3Promega Biosciences LLC, 277 Granada Dr, San Luis Obispo, CA 93401
Report abuse »
Questions
Ask the author a question about this poster.
Ask a Question »

Creative Commons

Related Posters


riboPOOL: Affordable Custom/Ribosomal RNA Depletion Against Any Species for RNA-Seq
Catherine Goh1, Andrew Walsh1, Michaela Beitzinger1, Jonas Bertram1, Kristina Döring3, Sascha Dietrich3, Iana Kim4, Stefan Kotschote2, Claus Kuhn4, Konrad Förstner3, Michael Bonin2, Stefan Hannus1, Michael Hannus1

The Good, the Bad and the Ugly: Selective single cell isolation
Sandra Lubos1,2, Nils Körber1, Heide Marie Resch1, Iris Augustin2, Stefan Niehren1

Single-cell studies: Focus on reverse transcription
Zucha Daniel, Androvic Peter, Kubista Mikael, Valihrach Lukas

Osteolytic jaw lesions
Dr. Amal Hassan Zakarya, Sarah Ahmed Mohamed, Noura Mohamed Kamal, Heba Ahmed Saleh, Ali Abd Elwahed

Potential therapeutic effects of induced pluripotent stem cells on induced salivary gland cancer in experimental rats
Yasmine Alaa El-Din1, Dina Sabry2, Amal Hassan Abdelrahman3, and Safa Fathy3