Simultaneous high-resolution detection of multiple transcripts combined with localization of proteins in whole-mount embryos.
Simultaneous high-resolution detection of multiple transcripts combined with localization of proteins in whole-mount embryos.
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DOI：
10.1186/s12915-014-0055-7
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发表时间：
2014-08-15
期刊：
影响因子：
5.4
通讯作者：
Raz E
中科院分区：
文献类型：
--
作者：
Gross-Thebing T;Paksa A;Raz E
整体原位杂交（WISH）是研究完整胚胎和组织中RNA分子时空表达模式的基本工具。现有的胚胎mRNA检测方法依赖于产生可扩散产物的酶活性和化学反应，这些产物不与被检测的RNA固定，从而降低了该技术的空间分辨率。此外，目前的WISH技术耗时，且通常不与报告蛋白质分子表达的方法相结合。
我们在此开发并介绍的实验方案基于RNAscope技术，该技术目前用于福尔马林固定、石蜡包埋和冷冻组织切片的研究和临床应用。以斑马鱼胚胎为例，我们提供了一种稳健且快速的方法，能够同时显示多种转录本，在此展示了三种不同的RNA分子。优化后的程序能够保持胚胎的完整性，同时具有出色的信噪比。因此，采用这种方法能够在发育中的胚胎或所研究组织的三维环境中，高分辨率地确定多种RNA分子相互之间的空间表达模式和亚细胞定位。最后，我们表明该方法保留了在特定细胞或细胞器中表达的荧光蛋白的功能，并保持了抗原性，从而能够使用抗体进行蛋白质检测。
通过对RNAscope技术进行微调，我们成功地重新设计了适用于整体胚胎样本的实验方案。将这种稳健的方法用于斑马鱼并推广到其他生物，将对发育生物学、分子生物学和细胞生物学的研究产生重大影响。将该方法应用于整体临床样本也具有类似的重要意义。这样的实验方案将通过提供有关临床标志物三维表达模式的信息，为生物医学研究和临床诊断做出贡献。
本文的网络版（doi:10.1186/s12915 - 014 - 0055 - 7）包含补充材料，授权用户可获取。
Whole-mount in situ hybridization (WISH) is a fundamental tool for studying the spatio-temporal expression pattern of RNA molecules in intact embryos and tissues. The available methodologies for detecting mRNAs in embryos rely on enzymatic activities and chemical reactions that generate diffusible products, which are not fixed to the detected RNA, thereby reducing the spatial resolution of the technique. In addition, current WISH techniques are time-consuming and are usually not combined with methods reporting the expression of protein molecules. The protocol we have developed and present here is based on the RNAscope technology that is currently employed on formalin-fixed, paraffin-embedded and frozen tissue sections for research and clinical applications. By using zebrafish embryos as an example, we provide a robust and rapid method that allows the simultaneous visualization of multiple transcripts, demonstrated here for three different RNA molecules. The optimized procedure allows the preservation of embryo integrity, while exhibiting excellent signal-to-noise ratios. Employing this method thus allows the determination of the spatial expression pattern and subcellular localization of multiple RNA molecules relative to each other at high resolution, in the three-dimensional context of the developing embryo or tissue under investigation. Lastly, we show that this method preserves the function of fluorescent proteins that are expressed in specific cells or cellular organelles and conserves antigenicity, allowing protein detection using antibodies. By fine-tuning the RNAscope technology, we have successfully redesigned the protocol to be compatible with whole-mount embryo samples. Using this robust method for zebrafish and extending it to other organisms would have a strong impact on research in developmental, molecular and cell biology. Of similar significance would be the adaptation of the method to whole-mount clinical samples. Such a protocol would contribute to biomedical research and clinical diagnostics by providing information regarding the three-dimensional expression pattern of clinical markers. The online version of this article (doi:10.1186/s12915-014-0055-7) contains supplementary material, which is available to authorized users.
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影响因子：
--
作者：
Strasser MJ;Mackenzie NC;Dumstrei K;Nakkrasae LI;Stebler J;Raz E
通讯作者：
Raz E
影响因子：
3.7
作者：
Hanley, Mary Beth;Lomas, Woodrow;Park, Emily
通讯作者：
Park, Emily
影响因子：
2.4
作者：
Hauptmann, G;Gerster, T
通讯作者：
Gerster, T
影响因子：
7.2
作者：
Voronina, Ekaterina;Seydoux, Geraldine;Nagamori, Ippei
通讯作者：
Nagamori, Ippei
DOI：
10.3791/1229
发表时间：
2009-03-25
期刊：
Journal of visualized experiments : JoVE
影响因子：
--
作者：
Brend, Tim;Holley, Scott A
通讯作者：
Holley, Scott A