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A microfluidic processor for gene expression profiling of single human embryonic stem cells

机译:用于单个人胚胎干细胞基因表达谱分析的微流控处理器

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摘要

The gene expression of human embryonic stem cells (hESC) is a critical aspect for understanding the normal and pathological development of human cells and tissues. Current bulk gene expression assays rely on RNA extracted from cell and tissue samples with various degree of cellular heterogeneity. These cell population averaging data are difficult to interpret, especially for the purpose of understanding the regulatory relationship of genes in the earliest phases of development and differentiation of individual cells. Here, we report a microfluidic approach that can extract total mRNA from individual single-cells and synthesize cDNA on the same device with high mRNA-to-cDNA efficiency. This feature makes large-scale single-cell gene expression profiling possible. Using this microfluidic device, we measured the absolute numbers of mRNA molecules of three genes (B2M, Nodal and Fzd4) in a single hESC. Our results indicate that gene expression data measured from cDNA of a cell population is not a good representation of the expression levels in individual single cells. Within the G0/G1 phase pluripotent hESC population, some individual cells did not express all of the 3 interrogated genes in detectable levels. Consequently, the relative expression levels, which are broadly used in gene expression studies, are very different between measurements from population cDNA and single-cell cDNA. The results underscore the importance of discrete single-cell analysis, and the advantages of a microfluidic approach in stem cell gene expression studies.
机译:人类胚胎干细胞(hESC)的基因表达是理解人类细胞和组织正常和病理发展的关键方面。当前的大量基因表达测定依赖于从具有不同程度的细胞异质性的细胞和组织样品中提取的RNA。这些细胞群体平均数据很难解释,特别是出于了解基因在单个细胞发育和分化的最早阶段的调节关系的目的。在这里,我们报告了一种微流体方法,该方法可以从单个单细胞提取总mRNA,并在同一设备上以高的mRNA到cDNA效率合成cDNA。此功能使大规模单细胞基因表达谱分析成为可能。使用此微流控设备,我们在单个hESC中测量了三个基因(B2M,Nodal和Fzd4)的mRNA分子的绝对数量。我们的结果表明,从细胞群体的cDNA测量的基因表达数据不能很好地表示单个细胞的表达水平。在G0 / G1期多能hESC群体中,某些单个细胞并未以可检测的水平表达所有3个被询问的基因。因此,在基因表达研究中广泛使用的相对表达水平在从群体cDNA和单细胞cDNA的测量之间有很大差异。结果强调了离散单细胞分析的重要性,以及微流体方法在干细胞基因表达研究中的优势。

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