Abstract
Advancements in imaging technologies, especially approaches that allow the imaging of single RNA molecules, have opened new avenues to understand RNA regulation, from synthesis to decay with high spatial and temporal resolution. Here, we describe a protocol for single-molecule fluorescent in situ hybridization (smFISH) using three different approaches for synthesizing the fluorescent probes. The three approaches described are commercially available probes, single-molecule inexpensive FISH (smiFISH), and in-house enzymatically labeled probes. These approaches offer technical and economic flexibility to meet the specific needs of an experiment. In addition, we provide a protocol to perform automated smFISH spot detection using the software FISH-quant.
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Acknowledgments
This work was funded by the National Institutes of Health (R35-GM133885) (JMT), a Predoctoral Training Grant in Molecular Biology (NIH-T32-GM008730) (RG) and the RNA Bioscience Initiative at the University of Colorado Anschutz Medical Campus (RG and JMT). It was further funded by the RNA Bioscience Initiative Summer Intern Program at the University of Colorado Anschutz Medical Campus (PTV).
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Arora, A., Goering, R., Velez, P.T., Taliaferro, J.M. (2022). Visualization and Quantification of Subcellular RNA Localization Using Single-Molecule RNA Fluorescence In Situ Hybridization. In: Dassi, E. (eds) Post-Transcriptional Gene Regulation. Methods in Molecular Biology, vol 2404. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-1851-6_13
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DOI: https://doi.org/10.1007/978-1-0716-1851-6_13
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Publisher Name: Humana, New York, NY
Print ISBN: 978-1-0716-1850-9
Online ISBN: 978-1-0716-1851-6
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