Multiplex Hextuple Luciferase Assaying

Authors

Alejandro Sarrion-Perdigones
Yezabel Gonzalez
Lyra Chang
Tatiana Gallego-Flores
Damian W Young
Koen J T Venken

Publication Date

1-1-2022

Journal

Methods in Molecular Biology

DOI

10.1038/s41467-022-29646-1

PMID

35414140

PMCID

PMC9005503

PubMedCentral® Posted Date

4-12-2022

PubMedCentral® Full Text Version

Post-print

Published Open-Access

yes

Keywords

Biological Assay, Luciferases, Promoter Regions, Geneti/c, Protein Binding, Transcription Factors, Luciferase, assay, multiplex, hextuple, orthogonal, cellular signaling pathway, cell culture, transfection, pathway perturbation, microplate reader

Abstract

We recently expanded the commonly used dual luciferase assaying method toward multiplex hextuple luciferase assaying, allowing monitoring the activity of five experimental pathways against one control at the same time. In doing so, while our expanded assay utilizes a total of six orthogonal luciferases instead of two, this assay, conveniently, still utilizes the well-established reagents and principles of the widely used dual luciferase assay. Three quenchable D-luciferin-consuming luciferases are measured after addition of D-Luciferin substrate, followed by quenching of their bioluminescence (BL) and the measurement of three coelenterazine (CTZ)-consuming luciferases after addition of CTZ substrate, all in the same vessel. Here, we provide detailed protocols on how to perform such multiplex hextuple luciferase assaying to monitor cellular signal processing upstream of five transcription factors and their corresponding transcription factor-binding motifs, using a constitutive promoter as normalization control. The first protocol is provided on how to perform cell culture in preparation toward genetic or pharmaceutical perturbations, as well as transfecting a multiplex hextuple luciferase reporter vector encoding all luciferase reporter units needed for multiplex hextuple luciferase assaying. The second protocol details on how to execute multiplex hextuple luciferase assaying using a microplate reader appropriately equipped to detect the different BLs emitted by all six luciferases. Finally, the third protocol provides details on analyzing, plotting, and interpreting the data obtained by the microplate reader.

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