On-Chip Separation and Analysis of RNA and DNA from Single Cells
On-Chip Separation and Analysis of RNA and DNA from Single Cells
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DOI：
10.1021/ac4040218
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发表时间：
2014-02-18
影响因子：
7.4
通讯作者：
Santiago, Juan G.
中科院分区：
文献类型：
--
作者：
Shintaku, Hirofumi;Nishikii, Hidekazu;Santiago, Juan G.
同时分析单细胞的RNA和DNA仍然是一个挑战，因为这些物种具有非常相似的物理和生化特性，并且可能相互交叉污染。本发明提供了一种芯片上系统，其能够选择性裂解单个活细胞，提取、聚焦和绝对定量细胞质RNA质量，并使用电裂解和等速电泳（ITP）将其与细胞核中的DNA物理分离。这种绝对定量无需酶促扩增即可在5分钟内完成。细胞核被保存，其DNA荧光信号可独立测量。我们使用单个小鼠淋巴细胞证明了该技术，为此我们提取了平均每个细胞14.1 pg的总RNA。我们还展示了RNA的绝对量和DNA的相对量之间的相关性分析，显示与细胞周期相关的异质性。该技术与DNA和RNA的分级分离以及各自的下游测定兼容。
The simultaneous analysis of RNA and DNA of single cells remains a challenge as these species have very similar physical and biochemical properties and can cross-contaminate each other. Presented is an on-chip system that enables selective lysing of single living cells, extraction, focusing, and absolute quantification of cytoplasmic RNA mass and its physical separation from DNA in the nucleus using electrical lysing and isotachophoresis (ITP). This absolute quantitation is performed without enzymatic amplification in less than 5 min. The nucleus is preserved, and its DNA fluorescence signal can be measured independently. We demonstrate the technique using single mouse lymphocyte cells, for which we extracted an average of 14.1 pg of total RNA per cell. We also demonstrate correlation analysis between the absolute amount of RNA and relative amount of DNA, showing heterogeneity associated with cell cycles. The technique is compatible with fractionation of DNA and RNA and with downstream assays of each.