Rahman Samir, Zenklusen Daniel
Département de Biochimie, Faculté de Médecine, Université de Montréal, Montréal, QC, Canada.
Methods Mol Biol. 2013;1042:33-46. doi: 10.1007/978-1-62703-526-2_3.
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.
调控基因表达是所有细胞系统的一项主要任务。RNA的产生和降解在此过程中起着关键作用，准确测量细胞内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化分析方法依赖于从大量细胞中提取RNA，将其从天然环境中取出，从而失去空间信息以及细胞间的变异性。在本章中，我们描述了一种荧光原位杂交（FISH）技术，该技术通过在单细胞中检测单个RNA来规避这个问题。该技术使用多个用有机染料进行荧光标记的单链短DNA探针，这些探针与固定细胞中的靶RNA杂交，从而能够在单细胞水平和单分子分辨率下对RNA进行定量和定位。此处描述的方案已针对酿酒酵母进行了优化。
