中文摘要：分析单个细胞的基因表达能分辨异质和罕见的细胞群，但现有的单细胞基因分析技术受到规模、易用性和成本的限制，还无法广泛使用。本研究提出了一种新型微滴一步式逆转录聚合酶链反应(RT-PCR)平台，并演示了通过单次实验，从10多万个单细胞中同时检出三个目标物，并快速输出结果。在定制的混合试剂中加入噬菌体T7基因2.5蛋白，克服细胞裂解液引起的抑制，并允许对封装在纳升液滴中的单细胞进行一步RT-PCR。利用概率反褶积方法分析指示基因表达的荧光信号，以解释环境RNA和细胞双峰，产生单细胞基因特征图谱，并预测异质样本中的细胞频率。还开发了一个模拟模型来指导实验设计，优化分析的准确性和精密度。使用体外转录和小鼠细胞系混合物，证明了本方法单RNA分子和罕见细胞群的检测频率为0.1%。该技术经济、灵敏、适应性强，为高通量单细胞分析提供了一个方便的平台，可广泛用于研究和临床。
外文摘要：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.
作者：Ma, J；Tran, G；Wan, AMD；Young, EWK；Kumacheva, E；Iscove, NN；Zandstra, PW
作者单位：Univ Toronto；Univ Hlth Networ；Univ British Columbia
期刊名称：SCIENTIFIC REPORTS
期刊影响因子：3.998
出版年份：2021
出版刊次：1
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