Institute of Biomedical Engineering, University of Toronto, Toronto, ON, M5S 3G9, Canada.
Department of Medical Biophysics, University of Toronto, Toronto, ON, M5G 1L7, Canada.
Sci Rep. 2021 Mar 24;11(1):6777. doi: 10.1038/s41598-021-86087-4.
Gene expression analysis of individual cells enables characterization of heterogeneous and rare cell populations, yet widespread implementation of existing single-cell gene analysis techniques has been hindered due to limitations in scale, ease, and cost. Here, we present a novel microdroplet-based, one-step reverse-transcriptase polymerase chain reaction (RT-PCR) platform and demonstrate the detection of three targets simultaneously in over 100,000 single cells in a single experiment with a rapid read-out. Our customized reagent cocktail incorporates the bacteriophage T7 gene 2.5 protein to overcome cell lysate-mediated inhibition and allows for one-step RT-PCR of single cells encapsulated in nanoliter droplets. Fluorescent signals indicative of gene expressions are analyzed using a probabilistic deconvolution method to account for ambient RNA and cell doublets and produce single-cell gene signature profiles, as well as predict cell frequencies within heterogeneous samples. We also developed a simulation model to guide experimental design and optimize the accuracy and precision of the assay. Using mixtures of in vitro transcripts and murine cell lines, we demonstrated the detection of single RNA molecules and rare cell populations at a frequency of 0.1%. This low cost, sensitive, and adaptable technique will provide an accessible platform for high throughput single-cell analysis and enable a wide range of research and clinical applications.
单细胞基因分析技术能够对异质和稀有细胞群体进行特征描述，然而，由于规模、简便性和成本方面的限制，这些技术的广泛应用受到了阻碍。在这里，我们提出了一种新颖的基于微滴的一步法逆转录聚合酶链式反应（RT-PCR）平台，并在单个实验中快速读取结果的情况下，证明了该平台能够同时检测超过 100,000 个单细胞中的三个靶标。我们定制的试剂混合物包含噬菌体 T7 基因 2.5 蛋白，以克服细胞裂解物介导的抑制作用，并允许在纳升级液滴中包封的单细胞进行一步法 RT-PCR。使用概率反卷积方法分析荧光信号，以说明环境 RNA 和细胞二聚体，并生成单细胞基因特征谱，以及预测异质样本中的细胞频率。我们还开发了一个模拟模型来指导实验设计，优化该测定的准确性和精密度。使用体外转录物和小鼠细胞系的混合物，我们证明了能够以 0.1%的频率检测单 RNA 分子和稀有细胞群体。这种低成本、灵敏且适应性强的技术将为高通量单细胞分析提供一个易于使用的平台，并能够实现广泛的研究和临床应用。
