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Fluorescence In Situ Hybridization of Small Non-Coding RNAs

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Small Non-Coding RNAs

Part of the book series: Methods in Molecular Biology ((MIMB,volume 2300))

Abstract

The determination of the cellular localization of a noncoding RNA (ncRNA) is highly helpful to decipher its function. RNA-FISH is a powerful method to detect specific RNAs in fixed cells. It allows both localization and quantification of RNA molecules within individual cells and tissues. Refined RNA-FISH methods have also been developed to determine RNA transcription and degradation rates. This chapter describes an RNA-FISH protocol that we developed to study the expression and localization of satellite III (SATIII) RNAs. This specific class of ncRNAs is expressed in response to various cellular stresses, including heat shock. The protocol is based on the use of a biotinylated LNA probe subsequently detected by a Streptavidin, Alexa Fluor® 488 conjugate. A protocol allowing efficient coupling of RNA-FISH and protein detection by immunofluorescence is also described as well as the bioinformatics pipeline, Substructure Analyzer, we recently developed to automate fluorescence signal analysis.

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Acknowledgments

V.V. and G.H. were supported by a graduate fellowship from the french Ministère Délégué à la Recherche et aux Technologies. This work was supported by grants from the European Alternative Splicing Network of Excellence (EURASNET, FP6 life sciences, genomics and biotechnology for health) and the European Associated Laboratory (LEA) on pre-mRNA splicing created by CNRS, UL, UM1, UM2 and Max Planck Institut. A. Metz and H. Kempf are acknowledged for helpful discussions. The PTIBC platform of UMS2008 is thanked for the access to the SP2 Confocal Microscope.

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Authors and Affiliations

  1. Université de Lorraine, CNRS, IMoPA, F-54000 Nancy, France

    Valentin Vautrot, Géraud Heckler, Christelle Aigueperse & Isabelle Behm-Ansmant

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  1. Valentin Vautrot
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  2. Géraud Heckler
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  3. Christelle Aigueperse
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  4. Isabelle Behm-Ansmant
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Correspondence to Isabelle Behm-Ansmant .

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  1. Université de Lorraine, CNRS, IMoPA, Nancy, France

    Mathieu Rederstorff

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Vautrot, V., Heckler, G., Aigueperse, C., Behm-Ansmant, I. (2021). Fluorescence In Situ Hybridization of Small Non-Coding RNAs. In: Rederstorff, M. (eds) Small Non-Coding RNAs. Methods in Molecular Biology, vol 2300. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-1386-3_8

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  • DOI: https://doi.org/10.1007/978-1-0716-1386-3_8

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  • Publisher Name: Humana, New York, NY

  • Print ISBN: 978-1-0716-1385-6

  • Online ISBN: 978-1-0716-1386-3

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