Microarray analysis of cytoplasmic versus whole cell RNA reveals a considerable number of missed and false positive mRNAs.
Microarray analysis of cytoplasmic versus whole cell RNA reveals a considerable number of missed and false positive mRNAs.
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DOI：
10.1261/rna.1677409
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发表时间：
2009-10
期刊：
影响因子：
4.5
通讯作者：
H. Trask;Richard Cowper-Sal·lari;M. Sartor;J. Gui;C. Heath;Janhavi Renuka;Azara-Jane Higgins;Peter C
中科院分区：
文献类型：
--
作者：
H. Trask;Richard Cowper-Sal·lari;M. Sartor;J. Gui;C. Heath;Janhavi Renuka;Azara-Jane Higgins;Peter C
无一例外，每一项已发表的用于确定真核生物中差异 mRNA 水平的微阵列研究都使用从整个细胞中提取的 RNA。据推测，在微阵列基因表达分析中使用全细胞 RNA 可以提供稳态 mRNA 的合法谱。标准标记方法和普遍认为 mRNA 几乎完全存在于细胞质中的教条导致人们长期以来认为核 RNA 的贡献可以忽略不计。我们报告说，纯细胞质 RNA 揭示了差异表达的 mRNA，否则使用全细胞 RNA 时无法检测到这些差异表达的 mRNA，并且核 RNA 的包含会扭曲 mRNA 谱，从而产生大量假阳性，从而对全细胞基因表达微阵列结果产生很大影响。我们的结论是，为了生成稳定状态 mRNA 群体的有效图谱，必须排除核成分，并且为了获得细胞基因表达图谱的更真实视图，应分别分析核和细胞质 RNA 部分。
With no known exceptions, every published microarray study to determine differential mRNA levels in eukaryotes used RNA extracted from whole cells. It is assumed that the use of whole cell RNA in microarray gene expression analysis provides a legitimate profile of steady-state mRNA. Standard labeling methods and the prevailing dogma that mRNA resides almost exclusively in the cytoplasm has led to the long-standing belief that the nuclear RNA contribution is negligible. We report that unadulterated cytoplasmic RNA uncovers differentially expressed mRNAs that otherwise would not have been detected when using whole cell RNA and that the inclusion of nuclear RNA has a large impact on whole cell gene expression microarray results by distorting the mRNA profile to the extent that a substantial number of false positives are generated. We conclude that to produce a valid profile of the steady-state mRNA population, the nuclear component must be excluded, and to arrive at a more realistic view of a cell's gene expression profile, the nuclear and cytoplasmic RNA fractions should be analyzed separately.