Extensible Multiplex Real-time PCR of MicroRNA Using Microparticles

Area of Science:

• Molecular Biology
• Biotechnology
• Genomics

Background:

• Multiplex quantitative real-time PCR (qPCR) is crucial for analyzing multiple genetic targets in single phenotypes.
• Existing methods face challenges with the increasing number of genetic targets.
• Analysis of microRNA (miRNA) expression is vital for understanding gene regulation.

Purpose of the Study:

• To develop a readily extensible qPCR method for simultaneous expression analysis of multiple miRNA targets.
• To utilize microparticles with immobilized primers as discrete reactors for high-throughput analysis.
• To enable quantitative analysis of miRNAs from limited biological samples, such as extracellular vesicles (EVs).

Main Methods:

• Development of a multiplex qPCR system using primer-immobilized microparticles (200–500 μm diameter) as discrete reactors.
• Particles are engraved with 2D codes for identification, enabling non-fluorescent encoding for real-time PCR signal acquisition.
• Assay involves tens of particles, each with different primers, allowing independent real-time amplification and quantitative analysis of multiple targets.

Main Results:

• Achieved high amplification efficiency (>95%) with amplicons accumulating reliably within the microparticles.
• Demonstrated high-fidelity signal acquisition in real-time PCR due to non-fluorescent encoding.
• Successfully analyzed 10 different miRNAs simultaneously from a limited amount of purified extracellular vesicles (EVs) in a single chamber.

Conclusions:

• The proposed multiplex qPCR method using microparticles offers a highly efficient and extensible platform for miRNA expression analysis.
• This technique allows for simultaneous quantification of multiple miRNA targets from limited samples, including EVs.
• The discrete reactor approach ensures reliable amplification and accurate quantitative information for each target.