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In Situ Hybridization of circRNAs in Cells and Tissues through BaseScope™ Strategy

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Circular RNAs

Part of the book series: Methods in Molecular Biology ((MIMB,volume 2765))

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Abstract

Imaging-based approaches are powerful strategies that nowadays have been largely used to gain insight into the function of different types of macromolecules. As for RNA, it is becoming clear how important is its intracellular localization for the control of proper cell differentiation and development and how its perturbation can be linked to several pathological states. This aspect is even more important if one thinks of highly polarized cells such as neurons.

In this chapter, we describe in detail an innovative RNA-FISH approach for the detection of circular RNAs (circRNAs), a recently discovered class of noncoding RNAs, which display different subcellular localizations and whose functions still largely remain to be elucidated. The detection of these molecules represents a great challenge, above all because they share most of their sequence with the corresponding linear counterparts, from which they differ only for the back-splicing junction (BSJ) originating from the circularization reaction. This implies the use of RNA-FISH probes capable of specifically binding the BSJ and avoiding the detection of the linear counterpart. This requirement imposes the design of probes on a very small region, which implies the risk of obtaining a low and undetectable signal. The BaseScope™ Assay RNA-FISH technology overpasses this problem since it is based on branched-DNA probes. With this approach it is possible to target a specific region of the RNA, even small such as a splicing junction, and at the same time to obtain a strong and well detectable signal. All this is possible thanks to subsequent series of probes that, starting from the first hybridization to the BSJ, build a branched tree of probes that greatly amplifies the signal. Here we provide a detailed step-by-step protocol of BaseScope™ RNA-FISH on circRNAs coupled with immunofluorescence, both in cells and tissues, and we address difficulties which may arise when using this methodology that depend on cell type, specific permeabilization, image acquisition, and post-acquisition analyses.

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Acknowledgments

The authors are supported by grants from ERC-2019-SyG 855923-ASTRA, AIRC IG 2019 Id. 23053, PRIN 2017 n. 2017P352Z4, H2020 Program “Sapienza Progetti Collaborativi.”

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Author notes

  1. Eleonora D’Ambra and Erika Vitiello contributed equally.

Authors and Affiliations

  1. Center for Life Nano- & Neuro-Science, Fondazione Istituto Italiano di Tecnologia (IIT), Rome, Italy

    Eleonora D’Ambra & Irene Bozzoni

  2. Center for Human Technology, Istituto Italiano di Tecnologia (IIT), Genoa, Italy

    Erika Vitiello & Irene Bozzoni

  3. Department of Biology and Biotechnologies “Charles Darwin”, Sapienza University of Rome, Rome, Italy

    Erika Vitiello, Tiziana Santini & Irene Bozzoni

Authors
  1. Eleonora D’Ambra
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  2. Erika Vitiello
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  3. Tiziana Santini
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  4. Irene Bozzoni
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Corresponding author

Correspondence to Irene Bozzoni .

Editor information

Editors and Affiliations

  1. Klaus Tschira Institute for Integrative Computational Cardiology, University Heidelberg, Heidelberg, Germany

    Christoph Dieterich

  2. Department of Cellular, Computational and Integrative Biology – CIBIO, University of Trento, Trento, Italy

    Marie-Laure Baudet

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D’Ambra, E., Vitiello, E., Santini, T., Bozzoni, I. (2024). In Situ Hybridization of circRNAs in Cells and Tissues through BaseScope™ Strategy. In: Dieterich, C., Baudet, ML. (eds) Circular RNAs. Methods in Molecular Biology, vol 2765. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-3678-7_4

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  • DOI: https://doi.org/10.1007/978-1-0716-3678-7_4

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  • Publisher Name: Humana, New York, NY

  • Print ISBN: 978-1-0716-3677-0

  • Online ISBN: 978-1-0716-3678-7

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