Analyzing mRNA expression using single mRNA resolution fluorescent in situ hybridization.
Analyzing mRNA expression using single mRNA resolution fluorescent in situ hybridization.
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DOI：
10.1016/s0076-6879(10)70026-4
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发表时间：
2010
影响因子：
--
通讯作者：
Singer, Robert H.
中科院分区：
文献类型：
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作者：
Zenklusen, Daniel;Singer, Robert H.
mRNA作为转录的产物和翻译的蓝图，是基因表达途径的主要中间产物。因此，在研究基因表达时，准确测定mRNA水平的能力是一个主要要求。mRNA也是不同调控步骤的靶标，发生在不同的亚细胞区室中。因此，为了理解基因表达调控的不同步骤，有必要分析单细胞背景下的mRNA，保持空间信息。在这里，我们描述了一个循序渐进的方案荧光原位杂交（FISH），允许检测单个酵母细胞中的单个mrna。该方法允许对单个细胞中的mRNA表达进行定量分析，允许通过简单计数mRNA进行“绝对”定量。它进一步使我们能够研究mRNA代谢的许多方面，从转录到加工，定位和mRNA降解。
As the product of transcription and the blueprint for translation, mRNA is the main intermediate product of the gene expression pathway. The ability to accurately determine mRNA levels is, therefore, a major requirement when studying gene expression. mRNA is also a target of different regulatory steps, occurring in different subcellular compartments. To understand the different steps of gene expression regulation, it is therefore essential to analyze mRNA in the context of a single cell, maintaining spatial information. Here, we describe a stepwise protocol for fluorescent in situ hybridization (FISH) that allows detection of individual mRNAs in single yeast cells. This method allows quantitative analysis of mRNA expression in single cells, permitting “absolute” quantification by simply counting mRNAs. It further allows us to study many aspects of mRNA metabolism, from transcription to processing, localization, and mRNA degradation.