Elsevier

Developmental Biology

Volume 434, Issue 1, 1 February 2018, Pages 15-23
Developmental Biology

A safer, urea-based in situ hybridization method improves detection of gene expression in diverse animal species

https://doi.org/10.1016/j.ydbio.2017.11.015Get rights and content
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Highlights

  • An alternative ISH protocol is proposed, substituting formamide with urea.

  • Specimen morphology appears to be better preserved.

  • Signal detection significantly improves, reducing non-specific staining.

  • The method, developed for C. hemisphaerica, was successful with additional species.

  • Less toxic and simple, it constitutes a useful addition to existing techniques.

Abstract

In situ hybridization is a widely employed technique allowing spatial visualization of gene expression in fixed specimens. It has greatly advanced our understanding of biological processes, including developmental regulation. In situ protocols are today routinely followed 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 acid sequence. This step is generally carried out at high temperatures and in a denaturing solution, called hybridization buffer, commonly containing 50% (v/v) formamide – a hazardous chemical. When applied to the soft-bodied hydrozoan medusa Clytia hemisphaerica, we found that this traditional hybridization approach was not fully satisfactory, causing extensive deterioration of morphology and tissue texture which compromised our observation and interpretation of results. We thus tested alternative solutions for in situ detection of gene expression and, 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. Our new protocol not only yielded better morphologies and tissue consistency, but also notably improved the resolution of the signal, allowing more precise localization of gene expression and reducing aspecific staining associated with problematic areas. Given the improved results and reduced manipulation risks, we tested the urea protocol on other metazoans, two brachiopod species (Novocrania anomala and Terebratalia transversa) and the priapulid worm Priapulus caudatus, obtaining a similar reduction of aspecific probe binding. Overall, substitution of formamide by urea during in situ hybridization offers a safer alternative, potentially of widespread use 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.

Keywords

In situ hybridization
Urea
Formamide
Clytia
Novocrania
Terebratalia
Priapulus

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1

Present address: Institut de Génomique Fonctionnelle de Lyon (IGFL), École Normale Supérieure de Lyon, CNRS UMR 5242 - INRA USC 1370, 69364 Lyon cedex 07, France.