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The analysis of the spatial expression patterns of genes is important for deciphering their functions. In situ hybridization provides insight into gene expression patterns at the cellular level. Here we describe a procedure for performing in situ hybridization on sections of paraffin-embedded tissue, including synthesis of labeled RNA probes, hybridization of the probes with target mRNAs, and immunological detection of the signals. The method enables evaluation of the expression patterns of genes in a variety of tissues of rice., (© 2025. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

A commonly employed technique in molecular biology to evaluate the temporal and spatial expression of a certain gene is in situ hybridization. This method is an effective strategy to construct synexpression groups, co-expressed genes acting in shared biological processes, and to find new members of genes engaged in the same signaling pathways to discover similar spatial and temporal expression patterns in zebrafish embryos. The major disadvantage of this method is that RNA probes can penetrate within 2 days of post-fertilization embryos, and therefore, in later developmental stages, the probe can only reach the surface tissues. Further application of the method in histological sections will be required for a complete and accurate gene expression investigation. However, this method is highly effective at late embryogenesis and early larval stages for observing gene expression in endodermal derivatives and sensory organs. RNA probes for in situ hybridization can be prepared through in vitro transcription from plasmids carrying specific promoter elements and mRNA-specific cDNA, or an alternative polymerase chain reaction (PCR) method can be used through PCR amplification. This chapter describes the procedures for detecting gene expression in zebrafish embryos using whole-mount RNA in situ hybridization., (© 2024. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

The Porifera are one of the best candidates as the sister group to all other metazoans. Studies on this phylum are therefore expected to shed light on the origin and early evolution of key animal features. Transcriptomic or genomic data acquired during the last 10 years have highlighted the conservation of most of the main genes and pathways involved in the development of the other metazoans. The next step is to determine how similar genetic tool boxes can result in widely dissimilar body plan organization, dynamics, and life histories. To answer these questions, three main axes of research are necessary: (1) conducting more gene expression studies; (2) developing knockdown protocols; and (3) reinterpreting sponge cell biology using modern tools. In this chapter we focus on the in situ hybridization (ISH) technique, needed to establish the spatiotemporal expression of genes, both on whole mount individuals and paraffin sections, and at different stages of development (adults, embryos, larvae, buds) of the homoscleromorph sponge Oscarella lobularis.

Circular RNAs (circRNAs) are covalently closed transcripts generated by back-splicing reaction. The lack of free ends endows these RNA molecules with high stability thus allowing them to accumulate in tissues and body fluids. They are widely expressed in most organisms, are modulated during development and display tissue-specific expression, resulting particularly enriched in the nervous system. Deregulation of circRNA expression has also been associated with several pathological conditions including neurological diseases and cancer.Here we present a Northern blot procedure that allows the analysis of the expression of bona fide circRNAs through the use of a digoxigenin-labeled RNA probe and the immunodetection of the signals.

In situ hybridization is a tool for evaluation of gene expression within tissues or single cells. This protocol describes optimized sensitive fluorescence detection of gene transcripts (mRNAs) in semithin sections of Schistosoma mansoni adult worms using specifically designed and labeled RNA probes. Due to improved methodologies in tissue preservation, sectioning, amplification of fluorescent signal, and prehybridization tissue treatment, it is possible to detect transcripts in the fine structures of schistosomes. The protocol is sensitive enough to detect very low abundance targets. This procedure is optimized for tissues derived from S. mansoni adult worms; however, it can be successfully applied to other trematode species.

Whole-mount in situ hybridization (WMISH) is a commonly used technique for visualizing the expression profile of mRNAs in embryos. Unlike traditional in situ hybridization techniques, which require thin tissue sections, the WMISH technique allows gene expression patterns to be assessed over the entire embryo and structure. Here, we describe the detailed procedural steps of WMISH, including probe production, embryo fixation and staining, and post-hybridization signal detection. Using this protocol, we visualized highly specific expression patterns of Sonic hedgehog and Bmp4 mRNAs in E12.5 mouse embryos.

In situ hybridization (ISH) is one of the fundamental methods in developmental biology and neurobiology. Their first ISH protocols were reported in 1969 (Gall and Pardue, Proc Natl AcadSci USA 63:378-83, 1969). Since several decades, ISH based on the specific hybridization of 100-2000 nucleotides long probes enabled the localization of DNA/RNA sequences in tissues and cells with high cellular resolution. But sometimes a limited sensitivity notably in mineralized tissues (Obernosterer et al., Nature Protocols 2:1508-14, 2007).Here we describe a recent improvement of in situ hybridization efficiency by applying nucleotide locked nucleic acid (LNA)-incorporated oligodeoxynucleotide probes (20 LNA/DNA nucleotide probes) essentially used for noncoding miRNA and messenger RNAs.

In situ hybridization is a commonly used technique using an antisense RNA probe to localize a specific RNA sequence on histological sections. This approach can visualize the expression pattern of a gene of interest in a portion of tissues. Here, we detail an optimized method for performing in situ hybridization on mouse paraffin sections using digoxigenin (DIG)-labeled RNA probes.

Generation of 3' single-stranded DNA (ssDNA) tails at the ends of a double-strand break (DSB) is essential to repair the break through accurate homology-mediated repair pathways. Several methods have been developed to measure ssDNA accumulation at a DSB in the budding yeast Saccharomyces cerevisiae. Here, we describe one of these assays, which is based on the inability of restriction enzymes to cleave ssDNA. Digestion of genomic DNA prepared at different time points after DSB generation leads to the formation of ssDNA fragments whose length increases as the 5' strand degradation proceeds beyond restriction sites. After the separation by electrophoresis on alkaline denaturing agarose gel, these ssDNA fragments can be visualized by hybridization with an RNA probe that anneals with the 3'-undegraded DSB strand. This assay allows a direct and comprehensive visualization of DSB end processing.

Over the years, in situ hybridization has been used for visualizing spatial gene expression patterns. Direct comparison of the expression patterns of different genes is useful for studying various biological activities in many situations. If we can distinguish signals derived from different probes in the same hybridization reaction, the localization of multiple gene transcripts can be detected simultaneously. In this chapter, we describe a two-color in situ hybridization procedure, which enables us to compare the expression patterns of two genes in a single tissue section. First, we explain how to prepare RNA probes and tissue samples. Then, we explain how to perform an in situ experiment, including pre-hybridization, hybridization, post-hybridization, and detection steps. A stepwise detection procedure is used to obtain the two color signals.

High-throughput screening with a loss-of-function strategy is a logical and efficient way to identify novel genes involved in biological processes of interest. In zebrafish, morpholinos have been developed as a convenient tool to knock down gene expression. Here, we describe procedures for systematic screening using morpholinos in zebrafish to identify novel deubiquitylases involved in convergent extension during gastrulation. In this example, we examine candidates based on embryonic morphology and molecular signals of whole mount in situ hybridization assay.