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.
摘要：
控制基因表达的固有随机过程导致单个细胞之间的异质性，强调单细胞研究的重要性。单分子荧光原位杂交（smFISH）的出现使得能够在单细胞水平进行基因表达分析，同时通过完整固定细胞中mRNA的可视化和定量来包括空间维度。通过结合smFISH与免疫荧光（IF），一种全面的方法有助于mRNAs和蛋白质的研究，以将基因表达谱与不同的细胞状态相关联。本章可作为smFISH-IF方案的综合指南，该方案针对出芽酵母酿酒酵母中的基因表达分析进行了优化。我们利用smFISH可视化α-微管蛋白IF推断的细胞周期过程中CLB2细胞周期蛋白的mRNA定位模式。
