Methodology for Whole Mount and Fluorescent RNA In Situ Hybridization in Echinoderms: Single, Double, and Beyond
Identifying the location of a specific RNA in a cell, tissue, or embryo is essential to understand its function. Here we use echinoderm embryos to demonstrate the power of fluorescence in situ RNA hybridizations to localize sites of specific RNA accumulat
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Introduction RNA sequencing has become a routine and powerful technology to identify genes and their activity. An investigator can isolate RNA, prepare a cDNA library, sequence the cognate RNAs within several days, and enjoy costs that are routinely decreasing. The advantage of this technique is sensitivity (theoretically one can detect a single RNA molecule), definitive identification (sequences of several hundred nucleotides allows definitive identification in most cases), quantitation (based on known amounts of spiked RNA in the sample, one can estimate absolute abundance of RNAs) and even differential gene expression (comparing two different samples or treatments of cells enables broad analysis of what RNAs are different between the cells). The software pipelines to quickly and effectively identify gene expression have steadily improved for ease of
David J. Carroll and Stephen A. Stricker (eds.), Developmental Biology of the Sea Urchin and Other Marine Invertebrates: Methods and Protocols, Methods in Molecular Biology, vol. 2219, https://doi.org/10.1007/978-1-0716-0974-3_12, © Springer Science+Business Media, LLC, part of Springer Nature 2021
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analysis, so relatively novice users are able to extract important results. The shortcoming of this approach for RNA analysis is that it does not reveal the site within the tissue, embryo, or cell where the RNA accumulates. Does the RNA accumulate uniformly in the tissue? Only in a few cells? Does the RNA stay in the nucleus, accumulate at the leading edge of a motile cell, or in the vegetal pole of the egg? Knowing that information is essential to understanding the functionality of the RNA and any products resulting from it (e.g., proteins). Therefore, identifying candidate mRNAs by sequencing, or by orthology to mRNAs with great interest, one may wish to institute an in situ RNA hybridization strategy. This chapter is intended to assist investigators on protocols, troubleshooting, and analysis of such protocols using echinoderm embryos and larvae. Having identified an mRNA of interest for hybridization enables definitive results within 2–2.5 weeks, from PCR of the initial candidate to imaging the hybridized cells. Alternatives to this approach for gene activity include genetically tagging a gene of interest with, for example, green fluorescent protein and imaging the time and place where that gene is active based on the GFP-reporter expression. This sort of technique is not yet available for echinoderms, making RNA hybridizations in situ a critical tool for gene expression analysis. Fifty years ago upon this writing, Pardue and Gall [1] published the first results of in situ hybridization (Fig. 1; for wonderful historical perspectives of these developments, see [3]; and a personal account by [4]. They immobilized a nucleus from a Xenopus oocyte on a microscope slide using an agar coating, denatured its DNA with alkaline pH, and hybridized the specimen with an rRNA probe that had been radioactively labeled by culturing Xenopus ce
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