Gene expression profiling of single cells on large-scale oligonucleotide arrays
Gene expression profiling of single cells on large-scale oligonucleotide arrays
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DOI：
10.1093/nar/gkl740
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发表时间：
2006-12-01
影响因子：
14.9
通讯作者：
Klein, Christoph A.
中科院分区：
文献类型：
--
作者：
Hartmann, Claudia H.;Klein, Christoph A.
在过去的十年中，通过对细胞群体的全局基因表达分析，获得了对各种刺激的细胞反应调节的重要见解。最近，从自然环境中分离出来的稀有细胞的特定细胞功能和潜在的调控网络转移到了关注的中心。然而，低细胞数仍然阻碍了生物医学研究中稀有离体材料的基因表达谱。因此，我们开发了一种强大的方法，用于高密度寡核苷酸阵列上的单细胞基因表达谱分析，具有良好的低丰度转录本覆盖率。用mRNA含量非常低的新鲜分离的单细胞（包括单个上皮细胞、成熟和未成熟树突细胞和造血干细胞）广泛测试该方案。定量PCR证实，基于PCR的全球扩增方法并没有改变转录本丰度的相对比例和无监督的层次聚类分析显示，一个单独的细胞的组织起源是正确的基因表达谱反映。此外，来自树突状细胞的基因表达数据表明，可以在单个细胞中监测细胞分化和通路激活。
Over the last decade, important insights into the regulation of cellular responses to various stimuli were gained by global gene expression analyses of cell populations. More recently, specific cell functions and underlying regulatory networks of rare cells isolated from their natural environment moved to the center of attention. However, low cell numbers still hinder gene expression profiling of rare ex vivo material in biomedical research. Therefore, we developed a robust method for gene expression profiling of single cells on high-density oligonucleotide arrays with excellent coverage of low abundance transcripts. The protocol was extensively tested with freshly isolated single cells of very low mRNA content including single epithelial, mature and immature dendritic cells and hematopoietic stem cells. Quantitative PCR confirmed that the PCR-based global amplification method did not change the relative ratios of transcript abundance and unsupervised hierarchical cluster analysis revealed that the histogenetic origin of an individual cell is correctly reflected by the gene expression profile. Moreover, the gene expression data from dendritic cells demonstrate that cellular differentiation and pathway activation can be monitored in individual cells.