Haemato-Oncology Research Unit, Division of Molecular Pathology, Division of Cancer Biology, The Institute of Cancer Research (ICR), Sutton, UK.
Nat Protoc. 2014 Mar;9(3):505-16. doi: 10.1038/nprot.2014.032. Epub 2014 Feb 6.
Symmetric cell divisions give rise to two sister cells that are identical to each other, whereas asymmetric divisions produce two sister cells with distinctive phenotypes. Although cell division symmetry is usually determined on the basis of a few markers or biological functions, the overall similarity between sister cells has not been thoroughly examined at a molecular level. Here we provide a protocol to separate sister embryonic stem cells (ESCs) and to conduct multiplexed gene expression analyses at the single-cell level by using 48 ESC genes. The procedure includes the dissection of dividing, paired sister cells by micromanipulation, followed by cell lysis, reverse transcription, gene-specific cDNA amplification and multiplexed quantitative PCR analyses. This protocol can be completed in 10 d, and it can be readily adapted to other cell types that are able to grow in suspension culture.
对称细胞分裂产生两个彼此完全相同的姐妹细胞，而非对称分裂产生两个具有不同表型的姐妹细胞。尽管细胞分裂的对称性通常基于少数几个标志物或生物学功能来确定，但在分子水平上尚未彻底检查姐妹细胞之间的整体相似性。在这里，我们提供了一种通过使用 48 个 ESC 基因分离姐妹胚胎干细胞 (ESC) 并进行单细胞水平的多重基因表达分析的方案。该程序包括通过显微操作分离正在分裂的配对姐妹细胞，然后进行细胞裂解、逆转录、基因特异性 cDNA 扩增和多重定量 PCR 分析。该方案可以在 10 天内完成，并且可以很容易地适用于能够在悬浮培养中生长的其他细胞类型。
