Single-cell RT-LAMP mRNA detection by integrated droplet sorting and merging
Single-cell RT-LAMP mRNA detection by integrated droplet sorting and merging
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DOI：
10.1039/c9lc00161a
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发表时间：
2019-07-21
期刊：
影响因子：
6.1
通讯作者：
Cai, Dawen
中科院分区：
文献类型：
--
作者：
Chung, Meng Ting;Kurabayashi, Katsuo;Cai, Dawen
单细胞分辨率的转录组学分析的最新进展以前所未有的分辨率揭示了生物样品中细胞间的异质性。将单个细胞分配在单个微滴中并收获每个细胞的mRNA分子用于下一代测序已被证明是以高通量对来自大量细胞的转录组进行谱分析的有效方法。然而，回收全转录组的测定在样品制备中是耗时的，并且需要昂贵的试剂和测序成本。许多生物医学应用，如病原体检测，更喜欢选择的基因的高灵敏度，可靠性和低成本的检测。在这里，我们提出了一种基于液滴的微流体平台，可以无缝地进行芯片上液滴分选和合并，从而能够在短时间内完成多步反应测定。通过将裂解缓冲液和反应物混合物依次添加到微滴反应器中，我们开发了一种新的单细胞逆转录环介导等温扩增（scRT-LAMP）工作流程，以在1小时内定量不同细胞类型中的特定mRNA表达水平。包括单细胞包封、分选、裂解、反应物添加和定量mRNA检测在内的全芯片工作流程为各种生物医学研究提供了快速、稳健和高通量的实验方法。
Recent advances in transcriptomic analysis at single-cell resolution reveal cell-to-cell heterogeneity in a biological sample with unprecedented resolution. Partitioning single cells in individual micro-droplets and harvesting each cell's mRNA molecules for next-generation sequencing has proven to be an effective method for profiling transcriptomes from a large number of cells at high throughput. However, the assays to recover the full transcriptomes are time-consuming in sample preparation and require expensive reagents and sequencing cost. Many biomedical applications, such as pathogen detection, prefer highly sensitive, reliable and low-cost detection of selected genes. Here, we present a droplet-based microfluidic platform that permits seamless on-chip droplet sorting and merging, which enables completing multi-step reaction assays within a short time. By sequentially adding lysis buffers and reactant mixtures to micro-droplet reactors, we developed a novel workflow of single-cell reverse transcription loop-mediated-isothermal amplification (scRT-LAMP) to quantify specific mRNA expression levels in different cell types within one hour. Including single cell encapsulation, sorting, lysing, reactant addition, and quantitative mRNA detection, the fully on-chip workflow provides a rapid, robust, and high-throughput experimental approach for a wide variety of biomedical studies.