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Anal Chem. 2018 Jan 16;90(2):1273-1279. doi: 10.1021/acs.analchem.7b04050. Epub 2018 Jan 3.

Single-Cell RT-PCR in Microfluidic Droplets with Integrated Chemical Lysis.

Author information

1
Department of Bioengineering and Therapeutic Sciences, University of California-San Francisco , San Francisco, California, United States.
2
California Institute for Quantitative Biosciences (QB3), University of California-Berkeley , Berkeley, California, United States.
3
Chan Zuckerberg Biohub , 499 Illinois Street, San Francisco, California, United States.

Abstract

Droplet microfluidics can identify and sort cells using digital reverse transcription polymerase chain reaction (RT-PCR) signals from individual cells. However, current methods require multiple microfabricated devices for enzymatic cell lysis and PCR reagent addition, making the process complex and prone to failure. Here, we describe a new approach that integrates all components into a single device. The method enables controlled exposure of isolated single cells to a high pH buffer, which lyses cells and inactivates reaction inhibitors but can be instantly neutralized with RT-PCR buffer. Using our chemical lysis approach, we distinguish individual cells' gene expression with data quality equivalent to more complex two-step workflows. Our system accepts cells and produces droplets ready for amplification, making single-cell droplet RT-PCR faster and more reliable.

PMID:
29256243
PMCID:
PMC5991602
DOI:
10.1021/acs.analchem.7b04050
[Indexed for MEDLINE]
Free PMC Article

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