Capture and enumeration of mRNA transcripts from single cells using a microfluidic device.
Capture and enumeration of mRNA transcripts from single cells using a microfluidic device.
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DOI：
10.1039/c5lc00445d
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发表时间：
2015-06
期刊：
影响因子：
6.1
通讯作者：
M. Walsh;Alexander P. Hsiao;Ho Suk Lee;Zhixia Liu;Xiaohua Huang
中科院分区：
文献类型：
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作者：
M. Walsh;Alexander P. Hsiao;Ho Suk Lee;Zhixia Liu;Xiaohua Huang
准确测量来自单细胞的RNA转录物将能够精确分类细胞类型和表征在正常细胞生理学和疾病中起关键作用的细胞群体中的异质性。作为实现这一目标的一个步骤，我们已经开发了一种微流体装置和方法，用于自动流体动力学捕获单个哺乳动物细胞，随后固定和数字计数从单个细胞释放的聚腺苷酸化mRNA分子。使用单分子荧光成像，我们已经证明，从单个HeLa细胞的多聚腺苷酸化的mRNA分子可以在几分钟内被捕获，通过杂交的聚脱氧胸苷寡核苷酸共价连接在玻璃表面上的设备。发现单个HeLa细胞中的总mRNA分子计数彼此显著不同。我们的技术开辟了使用单个集成的微流体装置以单分子灵敏度直接数字计数来自单细胞的RNA转录本的可能性。
Accurate measurement of RNA transcripts from single cells will enable the precise classification of cell types and characterization of the heterogeneity in cell populations that play key roles in normal cellular physiology and diseases. As a step towards this end, we have developed a microfluidic device and methods for automatic hydrodynamic capture of single mammalian cells and subsequent immobilization and digital counting of polyadenylated mRNA molecules released from the individual cells. Using single-molecule fluorescence imaging, we have demonstrated that polyadenylated mRNA molecules from single HeLa cells can be captured within minutes by hybridization to polydeoxyribothymidine oligonucleotides covalently attached on the glass surface in the device. The total mRNA molecule counts in the individual HeLa cells are found to vary significantly from one another. Our technology opens up the possibility of direct digital enumeration of RNA transcripts from single cells with single-molecule sensitivity using a single integrated microfluidic device.