Single-Molecule Resolution Fluorescent In Situ Hybridization (smFISH) in the Yeast S. cerevisiae
Single-Molecule Resolution Fluorescent In Situ Hybridization (smFISH) in the Yeast S. cerevisiae
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DOI：
10.1007/978-1-62703-526-2_3
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发表时间：
2013-01-01
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影响因子：
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通讯作者：
Zenklusen, Daniel
中科院分区：
文献类型：
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作者：
Rahman, Samir;Zenklusen, Daniel
调节基因表达是所有细胞系统的主要任务。RNA的产生和降解在这一过程中起着关键作用，准确测量细胞mRNA水平对于理解基因表达调控至关重要。研究基因表达的经典生物化学测定依赖于从大量细胞中提取RNA，将它们从其天然环境中取出，从而丢失空间信息以及细胞间变异性。在本章中，我们描述了一种荧光原位杂交（FISH）技术，通过检测单个细胞中的单个RNA来规避这个问题。该技术采用多个单链短DNA探针，用有机染料荧光标记，与固定细胞中的靶RNA杂交，允许在单细胞水平和单分子分辨率下定量和定位RNA。本文所述的方案已针对酵母S.啤酒。
Regulating gene expression is a major task for all cellular systems. RNA production and degradation plays a critical role in this process and accurately measuring cellular mRNA levels is essential to understanding gene expression regulation. Classical biochemical assays that study gene expression rely on extracting RNAs from large populations of cells, taking them out of their native context and thereby losing spatial information as well as cell-to-cell variability. In this chapter, we describe a fluorescent in situ hybridization (FISH) technique that circumvents this problem by detecting single RNAs in single cells. The technique employs multiple single-stranded short DNA probes fluorescently labeled with organic dyes that hybridize to target RNAs in fixed cells, allowing quantification and localization of RNAs at the single-cell level and at single-molecule resolution. The protocol described here has been optimized for the yeast S. cerevisiae.