即将举行的网络研讨会:实现无需离心的流式细胞术样品制备工作流程的标准化
目前,单细胞测序常见的应用是在肿瘤研究中。研究人员近期通过单细胞基因组扩增、测序和组装,从海洋样本中鉴定出一种单细胞细菌。
单细胞测序的兴起不仅是新概念炒作,相关领域也是如此。长期以来,由于技术瓶颈,许多生物学问题没有得到解决。现在突然有一种技术可以解决这些问题,性价比很高,导致这种技术的大量应用。
以单细胞转录组测序技术为例,主要包括以下四个步骤,其中重点是如何捕获/分选单细胞,这是决定单细胞检测成本和通量的关键步骤。
主要步骤:在细胞分选方法中,主要有特异性分选和非特异性分选两种方法。这两种方法之间的关系有点类似于定量(仅针对特定的靶基因)和转录组测序。通过特定标记选择特定的靶细胞,然后对靶细胞进行测序,因此特定选择方法通常具有较低的通量。
非特异性选择方法通常是高通量方法。一般是通过特定的技术从一个样本中随机捕获大量的细胞单体,然后对大量的细胞进行直接的平行测序,再从大量的单细胞数据中找到感兴趣的细胞类型,用于后续的分析。在逆转录扩增的过程中,每个细胞的cDNA都会连接上一个特定的接头序列,保证了后续混合测序后每个细胞的数据能够再次解析。该芯片可用于96个细胞的并行测序,降低了成本。
单细胞测序对目标组织细胞图谱进行全景扫描的单细胞测序,一般需要检测至少几千甚至几十万个细胞,但芯片通量只有96个细胞,这意味着多次芯片捕获,会增加芯片耗材的成本。因此,我们需要更高通量的技术。转录组测序是一种在单细胞水平上对mRNA进行高通量测序的新技术。通过在整体水平上研究单个细胞的基因表达,成功解决了细胞分子机制研究中的共性问题,如细胞异质性、细胞数量少、无法进行常规高通量测序等。
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