Isolation of RNA and the synthesis and amplification of cDNA from antigen-specific T cells for genome-wide expression analysis.
Isolation of RNA and the synthesis and amplification of cDNA from antigen-specific T cells for genome-wide expression analysis.
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DOI：
10.1007/978-1-62703-290-2_13
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发表时间：
2013-01-01
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影响因子：
--
通讯作者：
Haining, W Nicholas
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文献类型：
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作者：
Barnitz, R Anthony;Imam, Sabrina;Haining, W Nicholas
全基因组基因表达分析已成为研究不同分化状态的一种非常有效的常规工具。然而，对包含高水平异质性的细胞总数的检查，如免疫反应期间的CD8(+) T细胞总数，是有限的，因为复杂性阻碍了准确的解释。来自群体的基因表达特征代表了群体中所有细胞的平均值，这将平滑小亚群体中大的表达变化，并几乎消除任何小的变化。然而，在少数亚群中的小表达变化，如抗原特异性CD8(+) T细胞对感染的反应，可能会产生相关的生物学后果。虽然从小的细胞亚群中可以分离出非常有限的RNA，但现在有方法从这些有限的RNA中合成和扩增cDNA，以达到微阵列分析所需的足够数量。在这里，我们描述了一个完整的方案，从少量细胞中提取RNA，从RNA合成cDNA，并以无偏倚的方法扩增该cDNA。该方案是一个有用的工具，用于研究许多亚群的全基因组表达特征，这些亚群在数字上很小，但在免疫反应和体内平衡中很重要。
Genome-wide gene expression analysis has become a very powerful routine tool for the study of distinct differentiation states. However, the examination of total populations of cells that contain high levels of heterogeneity, such as the total CD8(+) T cell population during an immune response, is limited because that complexity hampers accurate interpretation. The gene expression signatures from populations represent the average of all cells within the populations, which will smooth out large expression changes within small subpopulations and virtually eliminate any small changes. However, small expression changes within a minor subpopulation, such as antigen-specific CD8(+) T cells responding to an infection, can have relevant biological consequences. Although very limited amounts of RNA can be isolated from small subpopulations of cells, there are now methods to synthesize and amplify cDNA from this limited RNA in sufficient quantities needed for microarray analysis. Here, we describe a complete protocol to extract RNA from small numbers of cells, synthesize cDNA from that RNA, and amplify that cDNA in an unbiased method. This protocol is a useful tool for the study of genome-wide expression signatures from many of the subpopulations that are numerically small but important in immune responses and homeostasis.