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Related Experiment Videos

In situ mRNA isolation from a microfluidic single-cell array using an external AFM nanoprobe.

Xuan Li1, Yinglei Tao, Do-Hyun Lee

  • 1Department of Biomedical Engineering, University of California, Irvine, CA 92697, USA. aplee@uci.edu.

Lab on a Chip
|April 13, 2017
PubMed
Summary

This study introduces a novel platform for in situ messenger RNA (mRNA) extraction from single cells in microfluidic arrays. The system enables precise quantification of gene expression, aiding in the study of cellular mechanisms and heterogeneity.

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Area of Science:

  • Biotechnology and Bioengineering
  • Molecular Biology
  • Cell Biology

Background:

  • Current microfluidic devices often limit external access for single-cell analysis after trapping.
  • Traditional mRNA extraction methods typically require cell lysis, which can be destructive.
  • Understanding cellular mechanisms and heterogeneity requires precise single-cell transcriptomic analysis.

Purpose of the Study:

  • To develop an in situ mRNA extraction platform for single-cell transcriptomic analysis.
  • To enable quantification of marker gene expression in individual cells within microfluidic arrays.
  • To overcome limitations of closed microfluidic systems and destructive lysis methods.

Main Methods:

  • Cells are individually trapped in microwell arrays sealed with a thin polydimethylsiloxane (PDMS) membrane.

Related Experiment Videos

  • A modified atomic force microscopy (AFM) probe, a dielectrophoretic nanotweezer (DENT), penetrates the membrane for mRNA extraction.
  • Dielectrophoresis is used by the DENT to extract mRNA molecules from single cells.

    • Main Results:

    • Quantified single-cellular expression levels of housekeeping genes in HeLa cells.
    • Demonstrated cell viability is maintained when alternating-current (AC) voltage is below 1.5 Vpp during probing.
    • Successfully performed in situ mRNA isolation and marker gene expression evaluation from a mixture of SK-BR-3 and U937 cells.

    Conclusions:

    • The integrated platform offers non-destructive, precise single-cell mRNA probing within sealed microfluidic systems.
    • This versatile tool facilitates upstream sample processing and downstream multifunctional analysis for biomedical research.
    • Enables in-depth analysis of cellular mechanisms and population heterogeneity through single-cell transcriptomics.