Optimized FISH methods for visualizing RNA localization properties in Drosophila and human tissues and cultured cells
Optimized FISH methods for visualizing RNA localization properties in Drosophila and human tissues and cultured cells
复制标题
DOI：
10.1016/j.ymeth.2017.06.027
复制
发表时间：
2017-08-15
期刊：
影响因子：
4.8
通讯作者：
Lecuyer，Eric
中科院分区：
文献类型：
--
作者：
Diot，Cedric;Chin，Ashley;Lecuyer，Eric
真核基因表达是由大量的调控步骤来协调的，以调节蛋白质编码和非编码RNA分子的各种家族的合成、成熟和命运。因此，确定RNA分子的亚细胞定位特性对于收集其功能和阐明转录后基因调控途径具有相当重要的意义。几十年来，荧光原位杂交（FISH）已构成了黄金标准技术，用于评估RNA的表达和分布特性的培养细胞，组织标本，和整个安装生物。最近，已经发表了旨在推进多重RNA-FISH实验的几种尝试。然而，这些程序既需要资金，又具有技术挑战性，而其全部潜力尚未得到开发。在这里，我们描述了一种优化的RNA-FISH方法，采用酪胺信号放大系统，鲁棒地提高分辨率和灵敏度需要探索RNA定位在果蝇胚胎，组织和常见的培养的人类和昆虫细胞系。概述了在96孔板格式中进行高通量分析的方法学细节和关键参数。
Eukaryotic gene expression is orchestrated by a large number of regulatory steps to modulate the synthesis, maturation and fate of various families of protein-coding and non-coding RNA molecules. Defining the subcellular localization properties of an RNA molecule is thus of considerable importance for gleaning its function(s) and for elucidating post-transcriptional gene regulation pathways. For decades, fluorescentIn Situhybridization (FISH) has constituted the gold-standard technique for assessing RNA expression and distribution properties in cultured cells, tissue specimens, and whole mount organisms. Recently, several attempts aimed at advancing multiplex RNA-FISH experiments have been published. However, these procedures are both financially demanding and technically challenging, while their full potential remains unexploited. Here we describe an optimized RNA-FISH method employing the Tyramide Signal Amplification system that robustly enhances resolution and sensitivity needed for exploring RNA localization inDrosophilaembryos, tissues and commonly cultured human and insect cell lines. Methodological details and key parameters are outlined for high-throughput analyses conducted in 96-well plate format.