Examining multiple cellular pathways at once using multiplex hextuple luciferase assaying

Sensitive simultaneous assessment of multiple signaling pathways within the same cells requires orthogonal reporters that can assay over large dynamic ranges. Luciferases have arisen as possible genetically encoded candidates due to their sensitivity, versatility, and cost-effectiveness. Here, we expanded luciferase multiplexing in post-lysis endpoint luciferase assays from two towards six. Light emissions are distinguished by a combination of distinct substrates and emission spectra deconvolution. Using synthetic assembly cloning, all six luciferase reporter units are stitched together into one plasmid; facilitating delivery of all reporter units through a process we named solotransfection, minimizing experimental errors. We engineered a multiplex hextuple luciferase assay to probe pathway fluxes through five transcriptional response elements against a control constitutive promoter. We were able to monitor the 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, as well as its broad application across different research fields.