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Proc Natl Acad Sci U S A. 2011 Aug 23;108(34):13999-4004. doi: 10.1073/pnas.1019446108. Epub 2011 Aug 1.

High-throughput microfluidic single-cell RT-qPCR.

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1
Centre for High-Throughput Biology, University of British Columbia, Vancouver, BC, Canada V6T 1Z4.

Abstract

A long-sought milestone in microfluidics research has been the development of integrated technology for scalable analysis of transcription in single cells. Here we present a fully integrated microfluidic device capable of performing high-precision RT-qPCR measurements of gene expression from hundreds of single cells per run. Our device executes all steps of single-cell processing, including cell capture, cell lysis, reverse transcription, and quantitative PCR. In addition to higher throughput and reduced cost, we show that nanoliter volume processing reduced measurement noise, increased sensitivity, and provided single nucleotide specificity. We apply this technology to 3,300 single-cell measurements of (i) miRNA expression in K562 cells, (ii) coregulation of a miRNA and one of its target transcripts during differentiation in embryonic stem cells, and (iii) single nucleotide variant detection in primary lobular breast cancer cells. The core functionality established here provides the foundation from which a variety of on-chip single-cell transcription analyses will be developed.

PMID:
21808033
PMCID:
PMC3161570
DOI:
10.1073/pnas.1019446108
[Indexed for MEDLINE]
Free PMC Article
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