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Related Concept Videos

Reporter Genes02:11

Reporter Genes

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Reporter genes are a type of protein-coding gene that are often tagged to a gene of interest. Once inside a target cell, reporter genes usually produce visually identifiable characteristics like fluorescence and luminescence when expressed along with the gene of interest. Thus, reporter genes “report” the presence or absence of genes of interest in an organism, determine the gene expression pattern, or track the physical location of a DNA segment or protein in the cell.
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DNA Microarrays02:34

DNA Microarrays

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Microarrays are high-throughput and relatively inexpensive assays that can be automated to analyze large quantities of data at a time. They are used in genome-wide studies to compare gene or protein expression under two varied conditions, such as healthy and diseased states. Microarrays consist of glass or silica slides on which probe molecules are covalently attached through surface functionalization. Most commonly, the slides are prepared through the chemisorption of silanes to silica...
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Related Experiment Video

Updated: May 28, 2025

High-throughput Functional Screening using a Homemade Dual-glow Luciferase Assay
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A Dual-Structured Chromogenic Enzyme Platform for a Rapid, Sensitive, Durable, and Precise Gene Expression Analysis.

Mu-Shen Chang1, Chia-Yi Lee1, Yu-Yen Chang2

  • 1PhD Program in Life Science, College of Life Science, Kaohsiung Medical University, Kaohsiung 807, Taiwan.

Analytical Chemistry
|February 12, 2025
PubMed
Summary

A new dual-structured chromogenic enzyme (DSCE) platform offers a cost-effective and stable alternative to the dual luciferase reporter (DLR) assay for gene expression analysis. This method provides enhanced signal durability and a broader dynamic range, making it a valuable tool for researchers.

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Area of Science:

  • Biochemistry
  • Molecular Biology
  • Enzyme Engineering

Background:

  • The dual luciferase reporter (DLR) assay is a standard for gene expression analysis, offering normalization capabilities.
  • DLR assays can detect subtle signaling changes but rely on expensive and unstable substrates.
  • Existing DLR substrates limit operational efficiency and increase costs.

Purpose of the Study:

  • To introduce a novel dual-structured chromogenic enzyme (DSCE) platform as a cost-effective and stable alternative to DLR assays.
  • To engineer and optimize horseradish peroxidase (HRP) for cell membrane tethering or extracellular secretion.
  • To compare the performance of the DSCE platform with DLR assays in terms of sensitivity, durability, and cost.

Main Methods:

  • Engineered horseradish peroxidase (HRP) into membrane-tethered (mHRP) and secreted (sHRP) forms.
  • Utilized chromogenic substrates ABTS and TMB for signal detection.
  • Optimized substrate and enzyme combinations for internal control and sample signal detection.
  • Compared DSCE platform performance against DLR assays for dynamic range and signal durability.

Main Results:

  • The DSCE platform demonstrated a broader dynamic range and superior signal durability compared to DLR assays.
  • Optimized configuration used sHRP with ABTS for internal control and mHRP with TMB for sample detection.
  • DSCE substrates were found to be approximately 30-fold less expensive than DLR substrates.
  • The DSCE platform provided rapid detection with signals durable for over 8 hours.

Conclusions:

  • The DSCE platform offers a cost-effective, stable, and highly sensitive alternative for gene expression analysis.
  • This platform maintains normalization capabilities while significantly reducing substrate costs.
  • DSCE technology provides a durable and efficient method for detecting downstream signaling changes.