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Specific distinguishing between miRNA and pre-miRNA by blocker displacement SMOS-qPCR
Guodong Zhao1, Yanmiao Dai2, Chenjing Xia2
1Zhejiang University of Technology, Zhejiang, Hangzhou, 310014, China; ZJUT Yinhu Research Institute of Innovation and Entrepreneurship, Zhejiang, Hangzhou, 311400, China; Suzhou Key Laboratory of Integrated Traditional Chinese and Western Medicine for Digestive Diseases, Department of Spleen and Stomach Diseases, Kunshan Hospital of Traditional Chinese Medicine, Affiliated Hospital of Yangzhou University, Kunshan, Jiangsu, 215300, China; State Key Laboratory of Digital Medical Engineering, School of Biological Science and Medical Engineering, Southeast University, Nanjing, 211189, China.
Abstract:
MicroRNAs (miRNAs) play crucial roles in various biological processes, and their dysregulation is associated with numerous diseases. Accurate quantification of mature miRNAs is essential for their use as biomarkers. However, distinguishing between mature miRNAs and pre-miRNAs remains challenging. This study introduces a novel method called Blocker Displacement SMOS-qPCR (BL-SMOS-qPCR) to enhance the differentiation between miRNAs and pre-miRNAs. The method employs a blocker sequence complementary to the 3' end of pre-miRNA cDNA, effectively competing with the Linker sequence and reducing non-specific amplification. We optimized various parameters, including blocker modification, length, concentration, and reaction temperature. Results showed that MGB-modified blockers at optimal concentrations significantly improved discrimination between miRNAs and pre-miRNAs, reducing pre-miRNA signals by approximately 3.2-fold. The BL-SMOS-qPCR maintained similar dynamic ranges (6˟108 to 6˟101 copies per reaction) and R2 values compared to the original SMOS-qPCR method across multiple miRNA targets. Furthermore, the new method successfully distinguished between normal controls and esophageal cancer patients in serum samples, demonstrating its effectiveness in clinical applications. This study provides a novel approach for precise miRNA quantification, addressing the challenges of differentiating between mature miRNAs and their precursors in complex biological samples.
Insights
A new method, Blocker Displacement SMOS-qPCR, accurately quantifies microRNAs (miRNAs) by distinguishing mature forms from precursors. This advance improves miRNA biomarker reliability for disease detection.
Area of Science:
- Molecular Biology
- Biochemistry
- Genetics
Background:
- MicroRNAs (miRNAs) are vital regulators of biological processes.
- Dysregulated miRNA expression is linked to various diseases.
- Accurate quantification of mature miRNAs is crucial for biomarker development, but differentiating them from pre-miRNAs is challenging.
Purpose of the Study:
- To develop a novel quantitative method for precise differentiation between mature microRNAs and pre-microRNAs.
- To enhance the specificity of miRNA detection in biological samples.
Main Methods:
- Introduction of Blocker Displacement SMOS-qPCR (BL-SMOS-qPCR).
- Utilizing a blocker sequence complementary to pre-miRNA cDNA to prevent non-specific amplification.
- Optimization of blocker modifications, length, concentration, and reaction temperature.
Main Results:
- MGB-modified blockers significantly improved discrimination between mature miRNAs and pre-miRNAs, reducing pre-miRNA signals by ~3.2-fold.
- BL-SMOS-qPCR demonstrated comparable dynamic ranges and R² values to the original SMOS-qPCR method.
- The method successfully differentiated between normal controls and esophageal cancer patients using serum samples.
Conclusions:
- BL-SMOS-qPCR offers a robust approach for precise miRNA quantification.
- This method effectively addresses the challenge of distinguishing mature miRNAs from precursors in complex samples.
- The technique shows promise for clinical applications in disease diagnostics.
