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High-throughput microfluidic single-cell RT-qPCR

机译:高通量微流体单细胞RT-qPCR

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摘要

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 tabular 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.
机译:微流体研究中一个长期寻求的里程碑是用于可扩展分析单细胞转录的集成技术的发展。在这里,我们提出了一种完全集成的微流控设备,该设备能够每次运行数百个单细胞来执行基因表达的高精度RT-qPCR测量。我们的设备执行单细胞处理的所有步骤,包括细胞捕获,细胞裂解,逆转录和定量PCR。除了更高的通量和降低的成本外,我们还表明,纳升体积处理可降低测量噪声,提高灵敏度并提供单核苷酸特异性。我们将该技术应用于(i)K562细胞中的miRNA表达,(ii)胚胎干细胞分化过程中miRNA及其靶标之一的共调节,以及(iii)原发性单核苷酸变异检测的3,300个单细胞测量板状乳腺癌细胞。此处建立的核心功能为开发各种片上单细胞转录分析提供了基础。

著录项

  • 来源
  • 作者单位

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

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

    Centre for High-Throughput Biology, University of British Columbia, Vancouver, BC, Canada V6T 1Z4,Department of Physics and Astronomy, University of British Columbia, Vancouver, BC, Canada V6T 1Z4;

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

    Centre for High-Throughput Biology, University of British Columbia, Vancouver, BC, Canada V6T 1Z4,Department of Chemical and Biological Engineering, University of British Columbia, Vancouver, BC, Canada V6T 1Z4,Michael Smith Laboratories, University of British Columbia, Vancouver, BC, Canada V6T 1Z4;

    Michael Smith Genome Sciences Centre, BC Cancer Agency, Vancouver, BC, Canada V5Z 4S6;

    Department of Chemical and Biological Engineering, University of British Columbia, Vancouver, BC, Canada V6T 1Z4,Michael Smith Laboratories, University of British Columbia, Vancouver, BC, Canada V6T 1Z4;

    Centre for Translational and Applied Genomics, Vancouver, BC, Canada V5Z 4E6,Department of Pathology, University of British Columbia, Vancouver, BC, Canada V6T 2B5;

    Centre for High-Throughput Biology, University of British Columbia, Vancouver, BC, Canada V6T 1Z4,Department of Physics and Astronomy, University of British Columbia, Vancouver, BC, Canada V6T 1Z4;

  • 收录信息 美国《科学引文索引》(SCI);美国《生物学医学文摘》(MEDLINE);美国《化学文摘》(CA);
  • 原文格式 PDF
  • 正文语种 eng
  • 中图分类
  • 关键词

    real-time PCR; lab on a chip; transcriptional noise; microRNA; single-cell diagnostics;

    机译:实时PCR;芯片实验室转录噪音微小RNA;单细胞诊断;

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