Issue 9, 2017

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

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

Supplementary files

Article information

Article type
Paper
Submitted
08 fev 2017
Accepted
03 apr 2017
First published
03 apr 2017

Lab Chip, 2017,17, 1635-1644

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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