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Related Experiment Videos

PABLO-QA: A sensitive assay for quantifying monophosphorylated RNA 5' ends.

Jamie Richards1,2, Joel G Belasco1,2

  • 1Skirball Institute of Biomolecular Medicine, New York University School of Medicine, New York, NY 10016, USA.

STAR Protocols
|April 18, 2022
PubMed
Summary

Monophosphorylated 5' ends trigger RNA degradation. PABLO-QA is a new assay to precisely quantify these RNA ends using ligation and RT-PCR, aiding transcript analysis.

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Area of Science:

  • Molecular Biology
  • RNA Biology
  • Biochemistry

Background:

  • 5' monophosphorylated RNA ends are critical triggers for RNA degradation across all organisms.
  • Understanding the prevalence of these ends is essential for controlling RNA stability and gene expression.

Purpose of the Study:

  • To introduce PABLO-QA (Phosphorylation Assay By Ligation of Oligonucleotides and Quantitative Amplification), a novel method for quantifying 5' monophosphorylated RNA ends.
  • To provide a sensitive, low-cost assay for analyzing RNA transcripts that are otherwise undetectable.

Main Methods:

  • PABLO-QA utilizes the ligation of an oligonucleotide to 5' monophosphorylated RNA ends.
  • Quantitative amplification via RT-PCR enables precise measurement of ligation efficiency.
  • Comparison with internal standards and controls ensures accurate quantification.
Keywords:
Gene ExpressionMolecular BiologyMolecular/Chemical Probes

Related Experiment Videos

Main Results:

  • The PABLO-QA assay accurately quantifies the percentage of specific RNA 5' ends that are monophosphorylated.
  • The method demonstrates sensitivity allowing analysis of previously undetectable transcripts.
  • The assay is described as low-cost and efficient.

Conclusions:

  • PABLO-QA offers a robust tool for precise quantification of 5' monophosphorylated RNA termini.
  • This method facilitates deeper insights into RNA degradation pathways and transcriptomic analysis.
  • The assay enables the study of RNA species previously beyond detection limits.