期刊
PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA
卷 108, 期 34, 页码 13999-14004出版社
NATL ACAD SCIENCES
DOI: 10.1073/pnas.1019446108
关键词
real-time PCR; lab on a chip; transcriptional noise; microRNA; single-cell diagnostics
资金
- Genome BC
- Genome Canada
- Western Diversification
- Canadian Institutes of Health Research (CIHR)
- Terry Fox Foundation
- Natural Sciences and Engineering Research Council (NSERC)
- Michael Smith Foundation for Health Research
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.
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