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Dual-Luciferase Assay System for the Quantification of Promoter Activity.

Patricia Ballester1, Cristina Ferrándiz2

  • 1Instituto de Biología Molecular y Celular de Plantas, Consejo Superior de Investigaciones Científicas - Universitat Politècnica de València, Valencia, Spain.

Methods in Molecular Biology (Clifton, N.J.)
|January 10, 2026
PubMed
Summary

Transient luciferase reporter assays offer sensitive plant gene regulation studies. The dual-luciferase system with Firefly luciferase (LUC) and Renilla luciferase (REN) improves accuracy for analyzing transcription factors and promoter activity.

Keywords:
AgroinfiltrationLuciferasePromoterTranscription factor (TF)

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Area of Science:

  • Plant molecular biology
  • Biotechnology
  • Genetics

Background:

  • Transient luciferase reporter assays are crucial for studying gene regulation in plants.
  • The dual-luciferase system enhances accuracy by normalizing reporter signals.
  • Agroinfiltration in Nicotiana benthamiana is a common method for transient gene expression.

Purpose of the Study:

  • To present a detailed protocol for dual-luciferase transient expression assays in plants.
  • To emphasize best practices for achieving robust and reproducible results.
  • To facilitate the analysis of promoter activity and gene regulation.

Main Methods:

  • Utilizing Firefly luciferase (LUC) as the primary reporter and Renilla luciferase (REN) as an internal control.
  • Employing agroinfiltration-mediated transient expression in Nicotiana benthamiana leaves.
  • Performing promoter dissection through mutagenesis, deletion mapping, and TF studies.

Main Results:

  • Demonstration of a sensitive and reproducible method for measuring transcriptional activity.
  • Validation of the dual-luciferase system for normalizing experimental variations.
  • Successful application in high-throughput analysis of transcriptional regulation.

Conclusions:

  • Dual-luciferase transient assays provide a powerful tool for plant promoter analysis.
  • The presented protocol ensures reliable investigation of gene regulatory mechanisms.
  • This method aids in the functional characterization of transcription factors and cis-regulatory elements.