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Detecting R-Loop Formation Using a Plasmid-Based In Vitro Transcription Assay.

Lei Shen1, Yanzhong Yang2

  • 1Department of Cancer Genetics and Epigenetics, Beckman Research Institute, City of Hope National Cancer Center, Duarte, CA, USA.

Methods in Molecular Biology (Clifton, N.J.)
|May 11, 2023
PubMed
Summary

This study presents a novel plasmid-based in vitro transcription assay to measure R-loop formation. This method, utilizing gel mobility and DRIP-qPCR, better reflects in vivo conditions by including DNA topology and transcription.

Keywords:
DNA/RNA immunoprecipitation-qPCRDot-blotGel electrophoresisIn vitro transcriptionR-loop

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Area of Science:

  • Molecular Biology
  • Genetics
  • Biochemistry

Background:

  • R-loops are critical three-stranded nucleic acid structures involved in gene expression and genome stability.
  • Understanding R-loop dynamics requires robust detection methods.

Purpose of the Study:

  • To describe a novel plasmid-based in vitro transcription assay for measuring R-loop formation.
  • To provide a method that more closely recapitulates in vivo conditions compared to existing assays.

Main Methods:

  • Plasmid-based in vitro transcription assay.
  • Detection and quantification using gel mobility, antibody staining, and DNA-RNA immunoprecipitation (DRIP)-qPCR.
  • Incorporation of DNA topology and active transcription as variables.

Main Results:

  • The described assay system effectively measures R-loop formation.
  • This method allows for the investigation of factors influencing R-loop dynamics.

Conclusions:

  • The developed assay provides a more physiologically relevant approach to study R-loops.
  • This method can be adapted to explore cis-elements and trans-acting factors affecting R-loop formation.