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Related Experiment Video

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Investigation of RNA Synthesis Using 5-Bromouridine Labelling and Immunoprecipitation

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Published on: May 3, 2018

A Radiolabeling-Free, qPCR-Based Method for Locus-Specific Pseudouridine Detection.

Zhixin Lei¹, Chengqi Yi²

¹Peking-Tsinghua Center for Life Sciences, Academy for Advanced Interdisciplinary Studies, Peking University, Beijing, 100871, China.

Angewandte Chemie (International Ed. in English)

|September 30, 2017

Summary

This study introduces a rapid, radiolabeling-free qPCR method for detecting pseudouridine (Ψ) in RNA. The facile technique enables sensitive detection of Ψ sites in various RNA types and aids in identifying pseudouridine synthases.

Keywords:

RNA modification epitranscriptomics gene expression high-resolution melting analysis nucleosides

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Area of Science:

Molecular Biology
Biochemistry
Genomics

Background:

Pseudouridine (Ψ) is the most abundant RNA modification.
Current locus-specific Ψ detection methods are often radiolabeled, complex, and time-consuming.

Purpose of the Study:

To develop a rapid, radiolabeling-free, qPCR-based method for locus-specific pseudouridine detection.
To enable sensitive detection of Ψ in low-abundance RNA and facilitate pseudouridine synthase identification.

Main Methods:

Chemical labeling of pseudouridine residues.
Reverse transcription (RT) inducing mutations/deletions at labeled sites.
qPCR with melting curve analysis to detect altered melting temperatures.

Main Results:

Validated the method on known Ψ sites in rRNA.
Successfully detected low-abundance Ψ in lncRNA and mRNA.
Identified PUS7-dependent Ψ616 in PSME2 mRNA.

Conclusions:

The developed method is facile, cost-effective, and rapid (1.5 days).
It offers sensitive detection of locus-specific pseudouridine.
The method is adaptable for detecting other epitranscriptomic modifications.