Legendre Félix, Cody Neal, Iampietro Carole, Bergalet Julie, Lefebvre Fabio Alexis, Moquin-Beaudry Gaël, Zhang Olivia, Wang Xiaofeng, Lécuyer Eric
Institut de Recherches Cliniques de Montréal.
J Vis Exp. 2013 Jan 30(71):e50057. doi: 10.3791/50057.
Assessing the expression pattern of a gene, as well as the subcellular localization properties of its transcribed RNA, are key features for understanding its biological function during development. RNA in situ hybridization (RNA-ISH) is a powerful method used for visualizing RNA distribution properties, be it at the organismal, cellular or subcellular levels. RNA-ISH is based on the hybridization of a labeled nucleic acid probe (e.g. antisense RNA, oligonucleotides) complementary to the sequence of an mRNA or a non-coding RNA target of interest. As the procedure requires primary sequence information alone to generate sequence-specific probes, it can be universally applied to a broad range of organisms and tissue specimens. Indeed, a number of large-scale ISH studies have been implemented to document gene expression and RNA localization dynamics in various model organisms, which has led to the establishment of important community resources. While a variety of probe labeling and detection strategies have been developed over the years, the combined usage of fluorescently-labeled detection reagents and enzymatic signal amplification steps offer significant enhancements in the sensitivity and resolution of the procedure. Here, we describe an optimized fluorescent in situ hybridization method (FISH) employing tyramide signal amplification (TSA) to visualize RNA expression and localization dynamics in staged Drosophila embryos. The procedure is carried out in 96-well PCR plate format, which greatly facilitates the simultaneous processing of large numbers of samples.
评估一个基因的表达模式及其转录RNA的亚细胞定位特性，是理解其在发育过程中生物学功能的关键特征。RNA原位杂交（RNA-ISH）是一种用于可视化RNA分布特性的强大方法，无论是在生物体、细胞还是亚细胞水平。RNA-ISH基于与感兴趣的mRNA或非编码RNA靶标序列互补的标记核酸探针（如反义RNA、寡核苷酸）的杂交。由于该程序仅需要一级序列信息来生成序列特异性探针，因此它可以普遍应用于广泛的生物体和组织标本。事实上，已经开展了许多大规模的ISH研究来记录各种模式生物中的基因表达和RNA定位动态，这导致了重要的社区资源的建立。虽然多年来已经开发了多种探针标记和检测策略，但荧光标记检测试剂和酶促信号放大步骤的联合使用显著提高了该程序的灵敏度和分辨率。在这里，我们描述了一种优化的荧光原位杂交方法（FISH），该方法采用酪胺信号放大（TSA）来可视化分期果蝇胚胎中的RNA表达和定位动态。该程序以96孔PCR板形式进行，这极大地便于同时处理大量样品。
