Department of Anatomy and Structural Biology, Albert Einstein College of Medicine, Bronx, NY, USA.
Systems Biology Lab, Amsterdam Institute of Molecular and Life Sciences (AIMMS), Vrije Universiteit Amsterdam, Amsterdam, The Netherlands.
Methods Mol Biol. 2020;2166:51-69. doi: 10.1007/978-1-0716-0712-1_4.
Single-molecule fluorescent in situ hybridization (smFISH) enables the detection and quantification of endogenous mRNAs within intact fixed cells. This method utilizes tens of singly labeled fluorescent DNA probes hybridized against the mRNA of interest, which can be detected by using standard wide-field fluorescence microscopy. This approach provides the means to generate absolute quantifications of gene expression within single cells, which can be used to link molecular fluctuations to phenotypes. To be able to correlate the expression of an mRNA and a protein of interest in individual cells, we combined smFISH with immunofluorescence (IF) in yeast cells. Here, we present our smFISH-IF protocol to visualize and quantify two cell cycle-controlled mRNAs (CLN2 and ASH1) and the cell cycle marker alpha-tubulin in S. cerevisiae. This protocol, which is performed over 2 days, can be used to visualize up to three colors at the time (i.e., two mRNAs, one protein). Even if the described protocol is designed for S. cerevisiae, we think that the considerations discussed here can be useful to develop and troubleshoot smFISH-IF protocols for other model organisms.
单分子荧光原位杂交 (smFISH) 可用于在完整固定细胞内检测和定量内源性 mRNAs。该方法利用数十个单独标记的荧光 DNA 探针与感兴趣的 mRNA 杂交，然后通过使用标准宽场荧光显微镜进行检测。这种方法提供了在单个细胞内生成基因表达绝对定量的手段，可用于将分子波动与表型联系起来。为了能够在单个细胞内关联感兴趣的 mRNA 和蛋白质的表达，我们将 smFISH 与酵母细胞中的免疫荧光 (IF) 结合使用。在这里，我们介绍了 smFISH-IF 方案，用于可视化和定量 S. cerevisiae 中的两个细胞周期控制的 mRNAs（CLN2 和 ASH1）和细胞周期标记物α-微管蛋白。该方案需要 2 天时间完成，一次最多可可视化三种颜色（即两种 mRNA，一种蛋白质）。即使该描述的方案是专为 S. cerevisiae 设计的，但我们认为这里讨论的注意事项对于为其他模式生物开发和解决 smFISH-IF 方案也会有所帮助。
