A Widely Applicable Urea-based Fluorescent/Colorimetric mRNA in situ Hybridization Protocol

Abstract

In situ hybridization methods are routinely employed to detect nucleic acid sequences, allowing to localize gene expression or to study chromosomal organization in their native context. These methods rely on the pairwise binding of a labeled probe to the target endogenous nucleic acid sequence-the hybridization step, followed by detection of annealed sequences by means of fluorescent or colorimetric reactions. Successful hybridization requires permeabilization of tissues, followed by denaturation of nucleic acids strands, which is usually carried out in a formamide-based buffer and at high temperatures. Such reaction conditions, besides posing a health hazard (both concerning manipulation and waste disposal), can be excessively harsh for the delicate tissues of some species or developmental stages. We detail here an alternative method for in situ hybridization, where the toxic formamide is replaced with a urea solution. This substitution improved both tissues preservation and signal-To-noise detection, in several animal species. The protocol described here, originally developed for the hydrozoan jellyfish Clytia hemisphaerica, provides guidelines for adapting formamide-based traditional protocols to the urea variant. Urea-based protocols have already been successfully applied to diverse invertebrate and vertebrate species, showing the ease of such a modification, and providing the scientific community with a promising, safer and versatile tool.