High-throughput microfluidic single-cell RT-qPCR
High-throughput microfluidic single-cell RT-qPCR
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DOI：
10.1073/pnas.1019446108
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发表时间：
2011-08-23
影响因子：
11.1
通讯作者：
Hansen, Carl L.
中科院分区：
文献类型：
--
作者：
White, Adam K.;VanInsberghe, Michael;Hansen, Carl L.
微流体研究中一个长期寻求的里程碑是开发可扩展的单细胞转录分析集成技术。在这里，我们提出了一个完全集成的微流体装置，能够进行高精度的RT-qPCR测量的基因表达从数百个单细胞每次运行。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。除了更高的吞吐量和降低成本，我们表明，纳升体积处理降低测量噪声，增加灵敏度，并提供单核苷酸特异性。我们将这项技术应用于3,300个单细胞测量，包括（i）K562细胞中的miRNA表达，（ii）胚胎干细胞分化过程中miRNA及其靶转录物之一的共调节，以及（iii）原发性小叶乳腺癌细胞中的单核苷酸变异检测。这里建立的核心功能为开发各种芯片上单细胞转录分析奠定了基础。
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.