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RNA-FISH to study regulatory RNA at the site of transcription

Soler, Marta, Boque Sastre, Raquel and Guil, Sonia 2017. RNA-FISH to study regulatory RNA at the site of transcription. In: Napoli, Sara ed. Promoter Associated RNA, Vol. 1543. Methods in Molecular Biology, Humana Press, pp. 221-229. (10.1007/978-1-4939-6716-2_12)

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The increasing role of all types of regulatory RNAs in the orchestration of cellular programs has enhanced the development of a variety of techniques that allow its precise detection, quantification, and functional scrutiny. Recent advances in imaging and fluoresecent in situ hybridization (FISH) methods have enabled the utilization of user-friendly protocols that provide highly sensitive and accurate detection of ribonucleic acid molecules at both the single cell and subcellular levels. We herein describe the approach originally developed by Stellaris®, in which the target RNA molecule is fluoresecently labeled with multiple tiled complementary probes each carrying a fluorophore, thus improving sensitivity and reducing the chance of false positives. We have applied this method to the detection of nascent RNAs that partake of special regulatory structures called R loops. Their growing role in active gene expression regulation (Aguilera and Garcia-Muse, Mol Cell 46:115–124, 2012; Ginno et al., Mol Cell 45:814–825, 2012; Sun et al., Science 340:619–621, 2013; Bhatia et al., Nature 511:362–365, 2014) imposes the use of a combination of in vivo and in vitro techniques for the detailed analysis of the transcripts involved. Therefore, their study is a good example to illustrate how RNA FISH, combined with transcriptional arrest and/or cell synchronization, permits localization and temporal characterization of potentially regulatory RNA sequences.

Item Type: Book Section
Date Type: Published Online
Status: Published
Schools: Biosciences
Publisher: Humana Press
ISBN: 9781493967148
ISSN: 1064-3745
Last Modified: 06 Mar 2020 15:55

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