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Real-time reverse transcription-polymerase chain reaction, or Real-time RT-PCR, is an analytical tool used to determine the expression level of target genes. The method involves converting mRNA to complementary DNA with the help of an enzyme known as reverse transcriptase, followed by the PCR amplification of the cDNA. These two processes can be performed simultaneously in a single tube or separately as a two-step reaction.
The real-time quantification of the number of amplified products is...

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A Rapid High-throughput Method for Mapping Ribonucleoproteins RNPs on Human pre-mRNA

A Rapid High-throughput Method for Mapping Ribonucleoproteins RNPs on Human pre-mRNA

Published on: December 2, 2009

One-pot ligation-PCR for universal RNA biomarker detection.

Jinding Liu¹, Zhixin Xie¹, Nini Li¹

¹Key Laboratory of Medicinal Chemistry and Molecular Diagnosis (Hebei University), Ministry of Education, Key Laboratory of Analytical Science and Technology of Hebei Province, College of Chemistry and Materials Science, Hebei University, Baoding, 071002, Hebei, P. R. China. yqcheng@hbu.edu.cn.

|February 16, 2026

Summary

This study introduces a novel one-pot ligation-PCR assay for RNA biomarker detection. The streamlined method enhances accuracy and reduces contamination risk compared to conventional techniques.

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Last Updated: Feb 17, 2026

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Published on: December 2, 2009

Highly Efficient Ligation of Small RNA Molecules for MicroRNA Quantitation by High-Throughput Sequencing

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Published on: November 18, 2014

Visual Detection of Multiple Nucleic Acids in a Capillary Array

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Published on: November 15, 2017

Area of Science:

Molecular Biology
Biochemistry
Genomics

Background:

Conventional RNA detection methods like reverse transcription-PCR (RT-PCR) and ligation-PCR have limitations.
RT-PCR struggles with complex RNA targets, while ligation-PCR involves multi-step processes increasing contamination risk.
Existing methods lack efficiency and precision for detecting RNA biomarkers, especially those with high sequence similarity.

Purpose of the Study:

To develop an improved, one-pot ligation-PCR assay for sensitive and specific RNA biomarker detection.
To overcome the limitations of conventional RT-PCR and two-step ligation-PCR workflows.
To create a streamlined, closed-tube system for RNA analysis, reducing handling and contamination.

Main Methods:

Developed a glyoxal-assisted one-pot ligation-PCR assay integrating probe ligation and PCR amplification.
Utilized thermally responsive glyoxal-caged primers for controlled primer activation during PCR cycling.
Validated the assay on mRNA splice variants and explored its application for microRNA detection.

Main Results:

The one-pot assay demonstrated sensitivity comparable to conventional two-step ligation-PCR.
Achieved significantly improved discrimination among closely related mRNA splice isoforms.
Successfully extended the strategy for microRNA detection, showcasing its versatility.

Conclusions:

The glyoxal-assisted one-pot ligation-PCR assay offers a simplified, robust, and efficient alternative for RNA biomarker detection.
This streamlined approach minimizes operational complexity and contamination risk.
The adaptable framework facilitates precise RNA analysis for various targets, including splice variants and microRNAs.