Examining multiple cellular pathways at once using multiplex hextuple luciferase assaying

Abstract

Sensitive simultaneous assessment of multiple signaling pathways within the same cells requires orthogonal reporters that can assay over large dynamic ranges. Luciferases are such genetically encoded candidates due to their sensitivity, versatility, and cost-effectiveness. We expand luciferase multiplexing in post-lysis endpoint luciferase assays from two to six. Light emissions are distinguished by a combination of distinct substrates and emission spectra deconvolution. All six luciferase reporter units are stitched together into one plasmid facilitating delivery of all reporter units through a process we termed solotransfection, minimizing experimental errors. We engineer a multiplex hextuple luciferase assay to probe pathway fluxes through five transcriptional response elements against a control constitutive promoter. We can monitor effects of siRNA, ligand, and chemical compound treatments on their target pathways along with the four other probed cellular pathways. We demonstrate the effectiveness and adaptiveness of multiplex luciferase assaying, and its broad application across different research fields.

Coumarin Luciferins and Mutant Luciferases for Robust Multicomponent Bioluminescence Imaging

“…29 Substrate unmixing and image processing algorithms can rapidly detect unique enzyme-substrate pairings within complex mixtures. 30,72 Multiple classes of orthogonal probes are available for multicomponent imaging via these methods, but only a few exhibit the necessary optical properties (NIR bioluminescence) for sensitive imaging. Given the unique structures and robust emission of Pecan/CouLuc-1-NMe2, we reasoned that this pair would be useful for multiplexing with other red-shifted probes.…”

A ratiometric dual-color luciferase reporter for fast characterization of transcriptional regulatory elements

“…We surveyed a panel of luciferases that emit green or red luminescence (Sarrion-Perdigones et al, 2019) by transiently expressing them in Nicotiana benthamiana leaves through agroinfiltration. We extracted total protein from leaf tissues and measured luciferase emission spectra and stability.…”

“…Variability commonly found between samples and experiments can be reduced using ratiometric assays with a second invariable reporter as an internal reference (Vazquez-Vilar et al, 2017). However, activity of these dual reporters is measured by different methods, or in sequential reactions with distinct kinetics (Sarrion-Perdigones et al, 2019;Vazquez-Vilar et al, 2017). Additionally, these assays often require sample homogenization and protein extraction, and are thus unpractical for high-throughput assays.…”

Glucocorticoid counteracts cellular mechanoresponses by LINC01569-dependent glucocorticoid receptor–mediated mRNA decay

“…TA-treated and stretched HSFBs were cotransfected with 400 ng of luciferase reporter vectors for either the 3′UTR or 5′UTR of the mechanosensor. Cells were then subjected to qRT-PCR to measure mRNA levels ( 65 ).…”