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Issue 9, 2017
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In situ mRNA isolation from a microfluidic single-cell array using an external AFM nanoprobe

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Abstract

We present an in situ mRNA extraction platform to quantify marker-genes' expression levels of single target cells within high-density microfluidic trapping arrays. This platform enables single-cell transcriptomic analysis to reveal in-depth information of cellular mechanisms and population heterogeneity. Although microfluidic technology enables the automation of single-cell sorting, trapping and identification, most developed microfluidic devices are closed off and prevent single-cell access by external analytical equipment. Besides, cell lysing is usually required for mRNA extraction. In our platform, cells are trapped individually in a microwell array sealed by a 1 μm-thick polydimethylsiloxane (PDMS) membrane, and a modified atomic force microscopy (AFM) probe—a dielectrophoretic nanotweezer (DENT)—penetrates through the membrane and extracts mRNA molecules from a single cell by dielectrophoresis. The single-cellular expression levels of 3 housekeeping genes from HeLa cells were analyzed quantitatively based on the quantification of the extracted mRNAs, and the probed cells remained viable when the applied alternating-current (AC) voltage was lower than 1.5 Vpp during mRNA probing. We also performed in situ mRNA isolation from a mixture of SK-BR-3 and U937 cells, mimicking a blood sample that underwent primary enrichment of circulating tumor cells (CTCs), and evaluated various marker-genes' expressions. This integrated platform combines the non-destructive and precise-control of a single-cell mRNA probe with sealed microfluidic systems' capability of upstream sample processing and downstream multifunctional analysis to enable a versatile and powerful tool for biomedical research.

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

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Supplementary files

Article information


Submitted
08 Feb 2017
Accepted
03 Apr 2017
First published
03 Apr 2017

Lab Chip, 2017,17, 1635-1644
Article type
Paper

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

X. Li, Y. Tao, D. Lee, H. K. Wickramasinghe and A. P. Lee, Lab Chip, 2017, 17, 1635 DOI: 10.1039/C7LC00133A

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