Abstract
Individual mRNA molecules can be imaged in fixed cells by hybridization with multiple, singly labeled oligonucleotide probes, followed by computational identification of fluorescent signals. This approach, called single-molecule RNA fluorescence in situ hybridization (smRNA FISH), allows subcellular localization and absolute quantification of RNA molecules in individual cells. Here, we describe a simple smRNA FISH protocol for two-color imaging of a circular RNA, CDR1as, simultaneously with an unrelated messenger RNA. The protocol can be adapted to circRNAs that coexist with overlapping, noncircular mRNA isoforms produced from the same genetic locus.
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Acknowledgments
This work was supported by the Deutsche Forschungsgemeinschaft [RA838/5-1 to N.R.] and the Berlin Institute of Health [CRG2aTP7 to N. R.]. We thank Arjun Raj and our BIMSB colleagues Alexander Loewer, Dhana Friedrich, Stefan Preibisch, and Marcel Schilling for discussions and advice.
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Kocks, C., Boltengagen, A., Piwecka, M., Rybak-Wolf, A., Rajewsky, N. (2018). Single-Molecule Fluorescence In Situ Hybridization (FISH) of Circular RNA CDR1as. In: Dieterich, C., Papantonis, A. (eds) Circular RNAs. Methods in Molecular Biology, vol 1724. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-7562-4_7
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DOI: https://doi.org/10.1007/978-1-4939-7562-4_7
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