Abstract
RNA-fluorescence in situ hybridization (RNA-FISH) is an essential and widely used tool for visualizing RNA molecules in intact cells. Recent advances have increased RNA-FISH sensitivity, signal detection efficiency, and throughput. However, detection of endogenous mRNA splice variants has been challenging due to the limits of visualization of RNA-FISH fluorescence signals and due to the limited number of RNA-FISH probes per target. HiFENS (high-throughput FISH detection of endogenous pre-mRNA splicing isoforms) is a method that enables visualization and relative quantification of mRNA splice variants at single-cell resolution in an automated high-throughput manner. HiFENS incorporates HCR (hybridization chain reaction) signal amplification strategies to enhance the fluorescence signal generated by low abundance transcripts or a small number of FISH probes targeting short stretches of RNA, such as single exons. The technique offers a significant advance in high-throughput FISH-based RNA detection and provides a powerful tool that can be used as a readout in functional genomics screens to discover and dissect cellular pathways regulating gene expression and alternative pre-mRNA splicing events.
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Shilo, A., Pegoraro, G., Misteli, T. (2024). High-Throughput RNA-HCR-FISH Detection of Endogenous Pre-mRNA Splice Variants. In: Haimovich, G. (eds) Fluorescence In Situ Hybridization (FISH). Methods in Molecular Biology, vol 2784. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-3766-1_9
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DOI: https://doi.org/10.1007/978-1-0716-3766-1_9
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