Department of Bioengineering and Therapeutic Sciences, University of California-San Francisco , San Francisco, California, United States.
California Institute for Quantitative Biosciences (QB3), University of California-Berkeley , Berkeley, California, United States.
Anal Chem. 2018 Jan 16;90(2):1273-1279. doi: 10.1021/acs.analchem.7b04050. Epub 2018 Jan 3.
Droplet microfluidics can identify and sort cells using digital reverse transcription polymerase chain reaction (RT-PCR) signals from individual cells. However, current methods require multiple microfabricated devices for enzymatic cell lysis and PCR reagent addition, making the process complex and prone to failure. Here, we describe a new approach that integrates all components into a single device. The method enables controlled exposure of isolated single cells to a high pH buffer, which lyses cells and inactivates reaction inhibitors but can be instantly neutralized with RT-PCR buffer. Using our chemical lysis approach, we distinguish individual cells' gene expression with data quality equivalent to more complex two-step workflows. Our system accepts cells and produces droplets ready for amplification, making single-cell droplet RT-PCR faster and more reliable.
液滴微流控技术可以通过单个细胞的数字逆转录聚合酶链反应 (RT-PCR) 信号来识别和分选细胞。然而，目前的方法需要多个微加工设备来进行酶解细胞和添加 PCR 试剂，使得过程复杂且容易失败。在这里，我们描述了一种将所有组件集成到单个设备中的新方法。该方法能够控制将分离的单个细胞暴露于高 pH 缓冲液中，该缓冲液可以裂解细胞并使反应抑制剂失活，但可以立即用 RT-PCR 缓冲液中和。使用我们的化学裂解方法，我们可以区分单个细胞的基因表达，其数据质量与更复杂的两步工作流程相当。我们的系统可以接受细胞并产生准备好进行扩增的液滴，从而使单细胞液滴 RT-PCR 更快、更可靠。
