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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
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.
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.
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.