Abstract
Robust RNA labeling and imaging methods that enable the understanding of cellular RNA biogenesis and function are highly desired. In this context, we describe a practical chemical labeling method based on a bioorthogonal reaction, namely, azide–alkyne cycloaddition reaction, which facilitates the fluorescence imaging of newly transcribed RNA in both fixed and live cells. This strategy involves the transfection of an azide-modified UTP analog (AMUTP) into mammalian cells, which gets specifically incorporated into RNA transcripts by RNA polymerases present inside the cells. Subsequent posttranscriptional click reaction between azide-labeled RNA transcripts and a fluorescent alkyne substrate enables the imaging of newly synthesized RNA in cells by confocal microscopy. Typically, 50 μM to 1 mM of AMUTP and a transfection time of 15–60 min produce significant fluorescence signal from labeled RNA transcripts in cells.
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Acknowledgments
This work was supported by the Department of Science and Technology, India, SERB grant (EMR/2014/000419) to S.G.S. and the Centre of Excellence in Epigenetics grant from the Department of Biotechnology, Government of India to S.G. The authors wish to thank Arun Tanpure, Progya Mukherjee, Soumitra Athavale, Rahul Jangid, and Ashwin Kelkar for discussion and help with work that has led to the optimization of these protocols.
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Sawant, A.A., Galande, S., Srivatsan, S.G. (2018). Imaging Newly Transcribed RNA in Cells by Using a Clickable Azide-Modified UTP Analog. In: Gaspar, I. (eds) RNA Detection. Methods in Molecular Biology, vol 1649. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-7213-5_24
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DOI: https://doi.org/10.1007/978-1-4939-7213-5_24
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