棘皮动物整体原位杂交和荧光 RNA 原位杂交方法：单标、双标及以上。
Methodology for Whole Mount and Fluorescent RNA In Situ Hybridization in Echinoderms: Single, Double, and Beyond.
机构信息
Department of Molecular and Cellular Biology and Biochemistry, Brown University, Providence, RI, USA.
Department of Biology and Evolution of Marine Organisms, Stazione Zoologica Anton Dohrn, Naples, Italy.
出版信息
Methods Mol Biol. 2021;2219:195-216. doi: 10.1007/978-1-0716-0974-3_12.
Abstract
Identifying the location of a specific RNA in a cell, tissue, or embryo is essential to understand its function. Here we use echinoderm embryos to demonstrate the power of fluorescence in situ RNA hybridizations to localize sites of specific RNA accumulation in whole mount embryo applications. We add to this technology the use of various probe-labeling technologies to colabel multiple RNAs in one application and we describe protocols for incorporating immunofluorescence approaches to maximize the information obtained in situ. We offer alternatives for these protocols and troubleshooting advice to identify steps in which the procedure may have failed. Overall, echinoderms are wonderfully suited for these technologies, and these protocols are applicable to a wide range of cells, tissues, and embryos.
摘要
确定特定 RNA 在细胞、组织或胚胎中的位置对于了解其功能至关重要。在这里，我们使用棘皮动物胚胎来证明荧光原位 RNA 杂交技术在整体胚胎应用中定位特定 RNA 积累位置的强大功能。我们将各种探针标记技术用于该技术中，以在一个应用中对多个 RNA 进行共标记，并描述了结合免疫荧光方法的方案，以最大限度地获取原位信息。我们为这些方案提供了替代方案和故障排除建议，以确定可能导致程序失败的步骤。总体而言，棘皮动物非常适合这些技术，并且这些方案适用于广泛的细胞、组织和胚胎。
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