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A Multiplexed Luciferase-based Screening Platform for Interrogating Cancer-associated Signal Transduction in Cultured Cells
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Multiplex Hextuple Luciferase Assaying.

Alejandro Sarrion-Perdigones1, Yezabel Gonzalez1, Lyra Chang2,3

  • 1Verna and Marrs McLean Department of Biochemistry and Molecular Biology, Baylor College of Medicine, Houston, TX, USA.

Methods in Molecular Biology (Clifton, N.J.)
|July 13, 2022
PubMed
Summary
This summary is machine-generated.

Researchers developed a multiplex hextuple luciferase assay to simultaneously monitor five signaling pathways and one control. This advanced method uses established dual luciferase assay principles with six luciferases for comprehensive cellular analysis.

Keywords:
AssayCell cultureCellular signaling pathwayHextupleLuciferaseMicroplate readerMultiplexOrthogonalPathway perturbationTransfection

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Area of Science:

  • Molecular Biology
  • Biochemistry
  • Cell Biology

Background:

  • The dual luciferase assay is a standard method for measuring gene expression.
  • Simultaneous monitoring of multiple signaling pathways is crucial for understanding complex cellular processes.
  • Existing methods often lack the capacity for high-throughput, multiplexed pathway analysis.

Purpose of the Study:

  • To expand the dual luciferase assay into a multiplex hextuple luciferase assay.
  • To enable simultaneous monitoring of five experimental pathways and one control pathway.
  • To provide detailed protocols for performing and analyzing multiplexed reporter assays.

Main Methods:

  • Utilized six orthogonal luciferases, categorized into D-luciferin-consuming and coelenterazine-consuming types.
  • Developed a sequential measurement strategy involving substrate addition, bioluminescence quenching, and subsequent substrate addition.
  • Established protocols for cell culture, reporter vector transfection, microplate reader-based detection, and data analysis.

Main Results:

  • Successfully implemented a hextuple luciferase assay capable of measuring six distinct bioluminescent signals in a single vessel.
  • Demonstrated the ability to monitor cellular signal processing upstream of five transcription factors concurrently.
  • Provided comprehensive protocols for the practical execution and interpretation of the assay.

Conclusions:

  • The multiplex hextuple luciferase assay offers a powerful tool for high-throughput pathway analysis.
  • This method simplifies the simultaneous assessment of multiple signaling events.
  • The detailed protocols facilitate the adoption and application of this advanced assay in various research settings.