Examining multiple cellular pathways at once using multiplex hextuple luciferase assaying

Nat Commun. 2019 Dec 13;10(1):5710. doi: 10.1038/s41467-019-13651-y.


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.

Publication types

  • Evaluation Study
  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't

MeSH terms

  • Biological Assay / methods*
  • Cell Line, Tumor
  • Genes, Reporter / genetics
  • Humans
  • Ligands
  • Luciferases / chemistry
  • Luciferases / genetics*
  • Luminescent Measurements / methods*
  • Plasmids / genetics
  • Promoter Regions, Genetic / genetics
  • Protein Engineering
  • RNA, Small Interfering / metabolism
  • Signal Transduction / drug effects
  • Signal Transduction / genetics


  • Ligands
  • RNA, Small Interfering
  • Luciferases