Single cell digital polymerase chain reaction on self-priming compartmentalization chip

Area of Science:

• Molecular Biology
• Biotechnology
• Genomics

Background:

• Single-cell analysis offers a powerful framework for understanding biological processes and diseases.
• Absolute quantification of single-cell gene expression remains challenging, hindering detailed biological insights.
• Existing microfluidic digital PCR devices have limitations in single-cell analysis and detection variability.

Purpose of the Study:

• To develop and validate a novel self-priming compartmentalization (SPC) microfluidic digital PCR chip.
• To enable absolute quantification of single-cell gene expression with high sensitivity and precision.
• To demonstrate the chip's utility in revealing single-cell heterogeneity for biological and disease studies.

Main Methods:

• Development of a power-free, valve-free SPC microfluidic digital PCR chip for single-molecule amplification.
• Optimization of protocols for high sample digitization rate (85%) and simultaneous detection of four single cells.
• Validation using β-actin DNA gene expression across different cell numbers and 18S rRNA reproducibility assessment.

Main Results:

• The SPC chip demonstrated high sensitivity, precision, and reproducibility in quantifying gene expression from single cells.
• Coefficient of variation for 18S rRNA detection in single cells ranged from 1.6% to 4.6%.
• PLAU gene expression heterogeneity in A549 lung cancer cells was successfully demonstrated at the single-cell level.

Conclusions:

• The developed SPC microfluidic digital PCR chip enables absolute quantification of single-cell gene copy numbers with enhanced sensitivity.
• The chip reduces labor time and reagent consumption, facilitating advanced single-cell studies.
• This technology is expected to significantly advance research in fundamental biology and disease mechanisms.