A safer, urea-based in situ hybridization method improves detection of gene expression in diverse animal species
A safer, urea-based in situ hybridization method improves detection of gene expression in diverse animal species
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一种更安全的基于尿素的原位杂交方法改善了不同动物物种基因表达的检测
DOI：
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发表时间：
2017
期刊：
影响因子：
--
通讯作者：
L. Leclère
中科院分区：
文献类型：
--
作者：
C. Sinigaglia;D. Thiel;A. Hejnol;E. Houliston;L. Leclère
原位杂交是一种广泛使用的技术，允许在固定标本中的基因表达的空间可视化。它已被证明是必不可少的，我们对生物过程的理解，包括发育调节。原位方案如今在许多实验室中是常规的，并且尽管细节可能改变，但它们都包括杂交步骤，其中特异性反义RNA或DNA探针退火到靶核酸链。该步骤通常在高温和变性溶液中进行，所述变性溶液为杂交缓冲液，通常含有50%（v/v）甲酰胺。一个重要的缺点是，热甲酰胺会造成重大的健康风险，因此必须非常小心地处理。我们被提示测试替代杂交解决方案的基因表达的水螅Clytia hemisphaerica，在传统的协议引起广泛的恶化杂交过程中的形态和纹理的水母原位检测，阻碍观察和结果的解释。受北方和南方印迹分析的优化方案的启发，我们在杂交缓冲液中用等体积的8 M尿素溶液代替50%甲酰胺。新方案产生了更好的组织形态和一致性，并且还显着提高了信号的分辨率，允许更精确地定位基因表达，以及减少非特异性位点的染色。鉴于改进的结果，使用毒性较小的杂交溶液，我们测试了一些其他后生动物的尿素协议：两个腕足类物种（Novocrania anomala和Terebratalia transversa）和蠕虫Priapulus caudatus，获得类似的减少非特异性探针结合。总的来说，在原位杂交中用尿素取代甲酰胺提供了更安全的替代方案，在研究、医疗和教学方面可能有用。我们鼓励其他工作者在他们的研究生物体上测试这种方法，并希望他们也将获得更好的样品保存，更精确的表达模式和更少的问题，由于非特异性染色，因为我们在这里报告的Clytia medusae和Novocrania和Terebratalia发育中的幼虫。
In situ hybridization is a widely employed technique allowing spatial visualization of gene expression in fixed specimens. It has proven to be essential to our understanding of biological processes, including developmental regulation. In situ protocols are today routine in numerous laboratories, and although details might change, they all include a hybridization step, where specific antisense RNA or DNA probes anneal to the target nucleic acids strand. This step, in general, is carried out at high temperatures and in a denaturing solution, the hybridization buffer, commonly containing 50% (v/v) formamide. An important drawback is that hot formamide poses a significant health risk and so must be handled with great care. We were prompted to test alternative hybridization solutions for in situ detection of gene expression in the medusa of the hydrozoan Clytia hemisphaerica, where traditional protocols caused extensive deterioration of the morphology and texture during hybridization, hindering observation and interpretation of results. Inspired by optimized protocols for Northern and Southern blot analysis, we substituted the 50% formamide with an equal volume of 8 M urea solution in the hybridization buffer. The new protocol yielded better morphologies and consistency of tissues, and also notably improved the resolution of the signal, allowing more precise localization of gene expression, as well as reduced staining at non-specific sites. Given the improved results using a less toxic hybridization solution, we tested the urea protocol on a number of other metazoans: two brachiopod species (Novocrania anomala and Terebratalia transversa) and the worm Priapulus caudatus, obtaining a similar reduction of aspecific probe binding. Overall, substitution of formamide by urea in in situ hybridization offers safer alternative protocols, potentially useful in research, medical and teaching contexts. We encourage other workers to test this approach on their study organisms, and hope that they will also obtain better sample preservation, more precise expression patterns and fewer problems due to aspecific staining, as we report here for Clytia medusae and Novocrania and Terebratalia developing larvae.
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DOI：
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发表时间：
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期刊：
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