Koshiba-Takeuchi Kazuko
Faculty of Life Sciences, Toyo University, Ora-gun, Gunma, Japan.
Methods Mol Biol. 2018;1752:123-131. doi: 10.1007/978-1-4939-7714-7_12.
In situ hybridization is defined as one of the most useful and powerful methods to know where genes (e.g., mRNA, ncRNA) of interest are expressed in tissues. Expression of mRNA can be detected as blue or dark purple signals though hybridization, immunoreaction and coloring steps. Genome-wide approaches in various model animals have been conducted thoroughly, and have led to new research areas aimed at uncovering novel gene functions in cell differentiation and development. To elucidate gene function, spatiotemporal gene expression analysis is very important. Here I describe protocols of whole-mount and section in situ hybridization, and emphasize the relevance of optimizing temperature, and sodium concentration, in hybridization buffer and substrate to improve signal.
原位杂交被定义为了解感兴趣的基因（如mRNA、非编码RNA）在组织中何处表达的最有用和最强大的方法之一。通过杂交、免疫反应和显色步骤，mRNA的表达可以被检测为蓝色或深紫色信号。在各种模式动物中已经全面开展了全基因组方法，并且催生了旨在揭示细胞分化和发育中新型基因功能的新研究领域。为了阐明基因功能，时空基因表达分析非常重要。在这里，我描述了整体原位杂交和切片原位杂交的实验方案，并强调了优化杂交缓冲液和底物中的温度和钠浓度以改善信号的相关性。
