High-quality RNA and DNA from flow cytometrically sorted human epithelial cells and tissues
High-quality RNA and DNA from flow cytometrically sorted human epithelial cells and tissues
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DOI：
10.2144/02324rr06
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发表时间：
2002-04-01
期刊：
影响因子：
2.7
通讯作者：
Rabinovitch, PS
中科院分区：
文献类型：
--
作者：
Barrett, MT;Glogovac, J;Rabinovitch, PS
微阵列技术使核酸序列和表达的综合分析成为可能。然而，呃，从人体组织的纯化肿瘤细胞群中高效获取适合阵列分析的高质量 RNA 和 DNA 的技术尚未得到很好的解决。显微切割可以富集各种肿瘤组织中存在的细胞群，但它不容易自动化或快速执行，并且在某些组织中，无法仅根据形态学标准轻易分离出感兴趣的细胞。在这里，我们描述了一种从原代组织的流式细胞仪纯化的整个上皮细胞中有效分离 RNA 和 DNA 的方案。水性试剂RNAlater(TM) 可保存RNA，允许通过流式分选对整个上皮细胞进行免疫标记和纯化，无需特殊的仪器准备以降低RNase 活性。我们使用实时 PCR 来确定流式分选后的 RNA 质量。适合表达和基因型分析的高质量 RNA 和 DNA 可以很容易地从流式细胞仪纯化的人体组织肿瘤细胞群中获得。
Microarray technologies have made possible comprehensive analyses of nucleic acid sequence and expression. However,er, the technology, to obtain efficiently high-quality RNA and DNA suitable for array analysis from purified populations of neoplastic cells from human tissues has not been well addressed. Microdissection can enrich for populations of cells present in various tumor tissues, but it is not easily automated or performed rapidly, and there are tissues in which cells of interest cannot be readily isolated based on morphologic criteria alone. Here we describe a protocol for efficient RNA and DNA isolation from flow cytometrically purified whole epithelial cells from primary tissue. The aqueous reagent, RNAlater(TM), which preserves RNA, allows immunolabeling and purification of whole epithelial cells by flow sorting without special instrument preparation to reduce RNase activity. We used real-time PCR to determine RNA quality after flow sorting. High-quality RNA and DNA suitable for expression and genotype analysis can be readily obtained from flow cytometrically purified populations of neoplastic cells from human tissiues.