Kim Sangjin, Vaidya Kavya
Department of Physics, University of Illinois at Urbana-Champaign; Center for the Physics of Living Cells, University of Illinois at Urbana-Champaign; Carl R. Woese Institute for Genomic Biology, University of Illinois at Urbana-Champaign; Center for Biophysics and Quantitative Biology, University of Illinois at Urbana-Champaign;
Department of Physics, University of Illinois at Urbana-Champaign; Center for the Physics of Living Cells, University of Illinois at Urbana-Champaign.
J Vis Exp. 2020 Jul 30(161). doi: 10.3791/61520.
Single-molecule fluorescence in situ hybridization (smFISH) allows for counting the absolute number of mRNAs in individual cells. Here, we describe an application of smFISH to measure the rates of transcription and mRNA degradation in Escherichia coli. As smFISH is based on fixed cells, we perform smFISH at multiple time points during a time-course experiment, i.e., when cells are undergoing synchronized changes upon induction or repression of gene expression. At each time point, sub-regions of an mRNA are spectrally distinguished to probe transcription elongation and premature termination. The outcome of this protocol also allows for analyzing intracellular localization of mRNAs and heterogeneity in mRNA copy numbers among cells. Using this protocol many samples (~50) can be processed within 8 h, like the amount of time needed for just a few samples. We discuss how to apply this protocol to study the transcription and degradation kinetics of different mRNAs in bacterial cells.
单分子荧光原位杂交（smFISH）能够对单个细胞中的mRNA绝对数量进行计数。在此，我们描述了smFISH在测量大肠杆菌中转录速率和mRNA降解方面的应用。由于smFISH基于固定细胞，我们在时间进程实验的多个时间点进行smFISH，即当细胞在基因表达诱导或抑制时经历同步变化时。在每个时间点，mRNA的子区域通过光谱进行区分，以探测转录延伸和过早终止。该方案的结果还允许分析mRNA的细胞内定位以及细胞间mRNA拷贝数的异质性。使用该方案，大约50个样本可以在8小时内处理完毕，所需时间与处理少量样本相当。我们讨论了如何应用该方案来研究细菌细胞中不同mRNA的转录和降解动力学。
