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. 2024:2784:45-58.
doi: 10.1007/978-1-0716-3766-1_3.

RNA and Protein Detection by Single-Molecule Fluorescent in Situ Hybridization (smFISH) Combined with Immunofluorescence in the Budding Yeast S. cerevisiae

Anna Maekiniemi  1 , Robert H Singer  2
Affiliations

RNA and Protein Detection by Single-Molecule Fluorescent in Situ Hybridization (smFISH) Combined with Immunofluorescence in the Budding Yeast S. cerevisiae

Anna Maekiniemi et al. Methods Mol Biol. 2024.

Abstract

The inherent stochastic processes governing gene expression give rise to heterogeneity across individual cells, highlighting the importance of single-cell studies. The emergence of single-molecule fluorescent in situ hybridization (smFISH) enabled gene expression analysis at the single-cell level while including the spatial dimension through the visualization and quantification of mRNAs in intact fixed cells. By combining smFISH with immunofluorescence (IF), a comprehensive approach takes shape facilitating the study of mRNAs and proteins to correlate gene expression profiles to different cellular states. This chapter serves as a comprehensive guide to a smFISH-IF protocol optimized for gene expression analysis in the budding yeast S. cerevisiae. We utilize smFISH to visualize the mRNA localization pattern of the CLB2 cyclin over the course of the cell cycle inferred by alpha-tubulin IF.

Keywords: Cell cycle; Gene expression; Immunofluorescence; Microscopy; RNA localization; Single-molecule; smFISH; smFISH-IF.

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