Optimized protocol for single-molecule RNA FISH to visualize gene expression in S. cerevisiae

Single-molecule RNA fluorescence in situ hybridization (smFISH) allows subcellular visualization, localization, and quantification of endogenous RNA molecules in fixed cells. The spatial and intensity information of each RNA can be used to distinguish mature from nascent transcripts inside each cell...

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Detalles Bibliográficos
Autores principales: Patel, Heta P., Brouwer, Ineke, Lenstra, Tineke L.
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Elsevier 2021
Materias:
Protocol
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8264745/
https://www.ncbi.nlm.nih.gov/pubmed/34278333
http://dx.doi.org/10.1016/j.xpro.2021.100647
Descripción
Sumario:Single-molecule RNA fluorescence in situ hybridization (smFISH) allows subcellular visualization, localization, and quantification of endogenous RNA molecules in fixed cells. The spatial and intensity information of each RNA can be used to distinguish mature from nascent transcripts inside each cell, revealing both past and instantaneous transcriptional activity. Here, we describe an optimized protocol for smFISH in Saccharomyces cerevisiae with optimized lyticase digestion time and hybrization steps for more homogenous results. For complete details on the use and execution of this protocol, please refer to Donovan et al. (2019).

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