In situ mRNA isolation from a microfluidic single-cell array using an external AFM nanoprobe
In situ mRNA isolation from a microfluidic single-cell array using an external AFM nanoprobe
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DOI：
10.1039/c7lc00133a
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发表时间：
2017-05-07
期刊：
影响因子：
6.1
通讯作者：
Lee, Abraham P.
中科院分区：
文献类型：
--
作者：
Li, Xuan;Tao, Yinglei;Lee, Abraham P.
我们提出了一个原位mRNA提取平台，用于定量高密度微流控捕获阵列中单个靶细胞的标记基因表达水平。该平台使单细胞转录组学分析能够揭示细胞机制和群体异质性的深入信息。虽然微流控技术能够实现单细胞分选、捕获和识别的自动化，但大多数开发的微流控设备都是封闭的，并阻止外部分析设备进入单细胞。此外，mRNA提取通常需要细胞裂解。在我们的平台中，细胞被单独捕获在微孔阵列密封的1 μ m厚的聚二甲基硅氧烷（PDMS）膜，和一个修改的原子力显微镜（AFM）探针-介电泳纳米镊子（DENT）-穿透膜和提取mRNA分子从一个单一的细胞介电泳。基于提取的mRNA的定量，定量分析来自HeLa细胞的3个管家基因的单细胞表达水平，并且当在mRNA探测期间施加的交流（AC）电压低于1. 5V-pp时，探测的细胞保持存活。我们还从SK-BR-3和U937细胞的混合物中进行原位mRNA分离，模拟经历循环肿瘤细胞（CTC）的初级富集的血液样品，并评估各种标记基因的表达。该集成平台将单细胞mRNA探针的非破坏性和精确控制与密封微流控系统的上游样品处理和下游多功能分析能力相结合，为生物医学研究提供了多功能和强大的工具。
We present an in situ mRNA extraction platform to quantify marker-genes' expression levels of single target cells within high-density microfluidic trapping arrays. This platform enables single-cell transcriptomic analysis to reveal in-depth information of cellular mechanisms and population heterogeneity. Although microfluidic technology enables the automation of single-cell sorting, trapping and identification, most developed microfluidic devices are closed off and prevent single-cell access by external analytical equipment. Besides, cell lysing is usually required for mRNA extraction. In our platform, cells are trapped individually in a microwell array sealed by a 1 mu m-thick polydimethylsiloxane (PDMS) membrane, and a modified atomic force microscopy (AFM) probe-a dielectrophoretic nanotweezer (DENT)-penetrates through the membrane and extracts mRNA molecules from a single cell by dielectrophoresis. The single-cellular expression levels of 3 housekeeping genes from HeLa cells were analyzed quantitatively based on the quantification of the extracted mRNAs, and the probed cells remained viable when the applied alternating-current (AC) voltage was lower than 1.5 V-pp during mRNA probing. We also performed in situ mRNA isolation from a mixture of SK-BR-3 and U937 cells, mimicking a blood sample that underwent primary enrichment of circulating tumor cells (CTCs), and evaluated various marker-genes' expressions. This integrated platform combines the non-destructive and precise-control of a single-cell mRNA probe with sealed microfluidic systems' capability of upstream sample processing and downstream multifunctional analysis to enable a versatile and powerful tool for biomedical research.