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Related Experiment Video

Updated: Dec 17, 2025

High Throughput MicroRNA Profiling: Optimized Multiplex qRT-PCR at Nanoliter Scale on the Fluidigm Dynamic ArrayTM IFCs
07:27

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In-particle stem-loop RT-qPCR for specific and multiplex microRNA profiling.

Seungwon Jung1, Won Jin Kim1, Bong Kyun Kim2

  • 1Center for Molecular Recognition Research, Materials and Life Science Research Division, Korea Institute of Science and Technology, Seoul, 02792, Republic of Korea; Applied Chemistry, Kyung Hee University, Yongin, 17104, Republic of Korea.

Biosensors & Bioelectronics
|June 23, 2020
PubMed
Summary

This study introduces a novel particle-based method for microRNA (miRNA) profiling using quantitative reverse transcription polymerase chain reaction (RT-qPCR). The technique enhances specificity and simplifies multiplex assays for accurate miRNA expression analysis.

Keywords:
HydrogelMultiplex assayQuantitative reverse-transcription PCRStem-loop reverse transcriptionmicroRNA

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Last Updated: Dec 17, 2025

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Area of Science:

  • Molecular Biology
  • Biotechnology
  • Genomics

Background:

  • MicroRNA (miRNA) profiling is crucial for understanding gene regulation.
  • Existing quantitative reverse transcription polymerase chain reaction (RT-qPCR) methods can be labor-intensive and prone to non-specific reactions.
  • Developing more efficient and selective miRNA detection methods is essential.

Purpose of the Study:

  • To develop a novel particle-based multiplex quantitative reverse transcription polymerase chain reaction (RT-qPCR) assay for microRNA (miRNA) profiling.
  • To improve the specificity, efficiency, and ease of use in multiplex miRNA detection.
  • To enable accurate quantification of miRNA expression levels.

Main Methods:

  • Chemically immobilizing stem-loop RT primers and forward primers for target miRNAs onto particles.
  • Performing target-specific cDNA synthesis using immobilized stem-loop RT primers.
  • Conducting quantitative polymerase chain reaction (qPCR) using immobilized forward primers in a single PCR process.
  • Utilizing multiple particles for each miRNA to achieve high-fidelity multiplex assays.

Main Results:

  • Achieved high selectivity (270:1) in differentiating mature miRNA from precursor.
  • Demonstrated excellent discrimination among homologous let-7 family members with <5% cross-reaction rates.
  • Successfully performed a five-plex miRNA profiling assay on total RNA with results consistent with conventional singleplex methods.
  • Reduced non-specific reactions and minimized labor for multiplex assays.

Conclusions:

  • The particle-based multiplex RT-qPCR assay offers a novel, highly selective, and efficient method for miRNA profiling.
  • This approach simplifies complex multiplex assays, reduces non-specific amplification, and improves overall assay performance.
  • The developed method provides a robust platform for accurate and high-throughput miRNA expression analysis.