Abstract
Visualization of single mRNA molecules in fixed cells can be achieved using single molecule fluorescent in situ hybridization (smFISH). This approach enables accurate quantification of mRNA numbers and localization at a single-cell level. To ensure reliable results using smFISH, it is critical to use fluorescent probes that are highly specific to their RNA target. To facilitate probe design, we have created anglerFISH, a user-friendly command-line based pipeline. In this chapter, we present how to perform a smFISH experiment using user-designed and labeled probes.
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Acknowledgments
The authors would like to thank all the members of the Chao lab and the Giorgetti lab for their support in establishing the probe designing and experimental pipeline. We thank Daniel Mateju for critically reading the manuscript. In addition, we thank the Facility for Advanced Imaging and Microscopy at FMI for help in image acquisition. Research in the Chao lab is funded by the Novartis Research Foundation, the Swiss National Science Foundation (310031A_182314) and the SNF NCCR RNA & Disease network. Research in the Giorgetti lab is funded by the Novartis Research Foundation and the European Research Council (ERC) under the European Union’s Horizon 2020 research and innovation (grant agreement no. 759366, ‘BioMeTre’).
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Piskadlo, E., Eichenberger, B.T., Giorgetti, L., Chao, J.A. (2022). Design, Labeling, and Application of Probes for RNA smFISH. In: Scheiffele, P., Mauger, O. (eds) Alternative Splicing. Methods in Molecular Biology, vol 2537. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-2521-7_10
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DOI: https://doi.org/10.1007/978-1-0716-2521-7_10
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