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
单细胞测序就是指获得单独细胞遗传物质的测序技术,是指对单独细胞水平上,对基因或转录组开展获取增加和高通量测序测序剖析。该技术可以揭露但个细胞特有的基因结构和基因的表达情况,包含结构变异、拷贝数基因变异、RNA表述水准等相关信息,使不一样细胞种类得到精准区别,并有利于专家在但细胞水准开展分子机制的探索。
为什么做单细胞测序?
针对多细胞微生物而言,在细胞的瓦解和分裂环节中,势必会慢慢造成或大或小的差别,即遗传物质的多样性。比如,同一肿瘤组织中的差异细胞含义不一样基因和转录组等遗传物质,就临床医学利用上来讲,这类差别或能影响某类治疗法对于该恶性肿瘤发挥的作用。
传统科学研究方法是在多细胞前提下进行检验,即一次解决不计其数个细胞。例如,当科研人员要想检验某一蛋白的表达量,常见的方式如WesternBlot等,所得到的信号值反映的问题其实就是遗传基因在人群细胞中的表达均值后水平,这就会造成不一样细胞间表述多样性数据的遗失。那如果应用单细胞测序方式,如流式的细胞术技术,在单细胞水准对荧光强度进行测量,就可完全反应同一个细胞群内不一样细胞的基因组测序和转录组等遗传物质的情况。
单细胞测序的完成方法与里程碑式,现阶段,单细胞测序的办法。
1、分开出单独细胞,单独搭建其测序百度文库,然后进行测序。现阶段用以细胞分离出来的典范技术包含:关键应用于细胞样品流式的细胞仪技术及其关键应用于组织切片样品莹光捕捉显微镜激光切割(LaserCapturemicrodissection,LCM)等(Figure2)[2]。这类方法简单粗暴,却也存有显而易见的相通缺点:低扩散系数,高投入——当所测单细胞总数比较低时,研究成果不能反映情况;不过随着所测总数提高,测序成本也会跟着呈线形提高。
2、根据标识(barcode)的单细胞鉴别,即给每个不同类型的细胞授予一段独一无二的DNA序列。于中后期混和测序时,可将带上同样barcode编码序列的核酸分子视作来源于同一个细胞,进而可以通过一次建库,测出百余上百个单细胞的数据再加以区别。不言而喻,此类方式相比前面一种,优点更高。
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