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Updated: Apr 16, 2026
Probe-based Real-time PCR Approaches for Quantitative Measurement of microRNAs
Published on: April 14, 2015
Quantification of mature microRNAs using pincer probes and real-time PCR amplification
Tinghua Huang1, Jun Yang2, Guopin Liu1
1Black Pig Research Institute, Yangtze University, Jingzhou, Hubei, China; College of Animal Science, Yangtze University, Jingzhou, Hubei, China.
Abstract:
The robust and reliable detection of small microRNAs (miRNAs) is important to understand the functional significance of miRNAs. Several methods can be used to quantify miRNAs. Selectively quantifying mature miRNAs among miRNA precursors, pri-miRNAs, and other miRNA-like sequences is challenging because of the short length of miRNAs. In this study, we developed a two-step miRNA quantification system based on pincer probe capture and real-time PCR amplification. The performance of the method was tested using synthetic mature miRNAs and clinical RNA samples. Results showed that the method demonstrated dynamic range of seven orders of magnitude and sensitivity of detection of hundreds of copies of miRNA molecules. The use of pincer probes allowed excellent discrimination of mature miRNAs from their precursors with five Cq (quantification cycle) values difference. The developed method also showed good discrimination of highly homologous family members with cross reaction less than 5%. The pincer probe-based approach is a potential alternative to currently used methods for mature miRNA quantification.
Insights
A new pincer probe method accurately quantifies mature microRNAs (miRNAs) and distinguishes them from precursors. This sensitive technique offers a reliable alternative for miRNA detection in research and clinical applications.
Area of Science:
- Molecular Biology
- Biochemistry
- Genetics
Background:
- Accurate quantification of microRNAs (miRNAs) is crucial for understanding their biological functions.
- Distinguishing mature miRNAs from precursors and similar sequences presents a significant analytical challenge due to their small size.
Purpose of the Study:
- To develop and validate a novel two-step system for the selective quantification of mature miRNAs.
- To assess the sensitivity, dynamic range, and specificity of the developed method.
Main Methods:
- A two-step quantification system combining pincer probe capture and real-time PCR amplification was designed.
- The method's performance was evaluated using synthetic mature miRNAs and clinical RNA samples.
Main Results:
- The developed method exhibited a broad dynamic range spanning seven orders of magnitude.
- High sensitivity was achieved, detecting as few as hundreds of miRNA molecules.
- Excellent discrimination between mature miRNAs and precursors (5 Cq difference) and high specificity against homologous sequences (<5% cross-reaction) were demonstrated.
Conclusions:
- The pincer probe-based approach provides a robust and sensitive method for mature miRNA quantification.
- This technique offers a promising alternative to existing methods for miRNA analysis in various biological and clinical settings.
