Methodology for Whole Mount and Fluorescent RNA In Situ Hybridization in Echinoderms: Single, Double, and Beyond.
Methodology for Whole Mount and Fluorescent RNA In Situ Hybridization in Echinoderms: Single, Double, and Beyond.
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DOI：
10.1007/978-1-0716-0974-3_12
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发表时间：
2021
期刊：
影响因子：
--
通讯作者：
Wessel GM
中科院分区：
文献类型：
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作者：
Perillo M;Paganos P;Spurrell M;Arnone MI;Wessel GM
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展示荧光原位RNA杂交在整个安装胚胎应用中定位特定RNA积累的位置的能力。我们在这项技术中增加了使用各种探针标记技术在一个应用中联合标记多个RNA，并描述了结合免疫荧光方法的协议，以最大限度地利用原位获得的信息。我们提供了这些协议的替代方案和故障排除建议，以确定该过程可能失败的步骤。总体而言，棘皮动物非常适合这些技术，这些协议适用于广泛的细胞、组织和胚胎。
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-labelling technologies to co-label 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.