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Quantification of Circular RNAs Using Digital Droplet PCR
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Capillary-based integrated digital PCR in picoliter droplets.

Jinyu Chen1, Zhaofeng Luo, Lin Li

  • 1Department of Thermal Science and Energy Engineering, University of Science and Technology of China, Hefei, 230027, China. heliqun@ustc.edu.cn.

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Summary

This study introduces an integrated capillary-based droplet digital polymerase chain reaction (ddPCR) system for precise absolute quantification. The novel system minimizes droplet loss and cross-contamination, improving rare event detection in diagnostics and research.

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Area of Science:

  • Biotechnology
  • Molecular Diagnostics
  • Analytical Chemistry

Background:

  • Droplet digital polymerase chain reaction (ddPCR) is increasingly used in diagnostics, but current systems suffer from manual droplet transfer, causing loss and contamination.
  • Existing chip-based ddPCR systems face challenges with droplet coalescence due to wall wetting and thermal oscillation, particularly impacting rare mutation quantification.
  • Accurate absolute quantification is crucial for applications like early cancer diagnostics and measuring cellular diversity.

Purpose of the Study:

  • To develop and validate a novel capillary-based integrated ddPCR system for accurate absolute quantification.
  • To overcome limitations of manual transfer and chip-based coalescence in existing ddPCR technologies.
  • To enable sensitive detection and quantification of rare genetic events.

Main Methods:

  • An integrated system was designed using a High-Performance Liquid Chromatography (HPLC) T-junction for droplet generation.
  • A long HPLC capillary connected the droplet generator to capillary-based thermocycler and cytometer modules.
  • The system's performance was validated through the absolute quantification of a lung cancer-specific gene (LunX).

Main Results:

  • The capillary-based integrated ddPCR system demonstrated excellent linearity (0.9988) across a wide range of concentrations.
  • The system achieved accurate absolute quantification, validated by the detection of the LunX gene.
  • Compared to quantitative polymerase chain reaction (qPCR), the integrated ddPCR showed superior detection limits and fold-change measurement capabilities.

Conclusions:

  • The proposed capillary-based integrated ddPCR system offers a robust solution for absolute quantification, addressing droplet loss and contamination issues.
  • This all-in-one ddPCR approach provides enhanced sensitivity for detecting rare events.
  • The system holds significant potential as a powerful tool for clinical diagnostics and academic research involving rare event analysis.