Voigt Franka, Eglinger Jan, Chao Jeffrey A
Friedrich Miescher Institute for Biomedical Research, Basel, Switzerland.
Methods Mol Biol. 2018;1649:373-384. doi: 10.1007/978-1-4939-7213-5_25.
Quantitative fluorescence microscopy techniques are frequently applied to answer fundamental biological questions. Single-molecule RNA imaging methods have enabled the direct observation of the initial steps of the mRNA life cycle in living cells, however, the dynamic mechanisms that regulate mRNA translation are still poorly understood. We have developed an RNA biosensor that can assess the translational state of individual mRNA transcripts with spatiotemporal resolution in living cells. In this chapter, we describe how to perform a TRICK (translating RNA imaging by coat protein knock-off) experiment and specifically focus on a detailed description of our image processing and data analysis procedure.
定量荧光显微镜技术经常被用于回答基本的生物学问题。单分子RNA成像方法能够直接观察活细胞中mRNA生命周期的初始步骤，然而，调节mRNA翻译的动态机制仍知之甚少。我们开发了一种RNA生物传感器，它可以在活细胞中以时空分辨率评估单个mRNA转录本的翻译状态。在本章中，我们描述了如何进行TRICK（通过衣壳蛋白敲除进行翻译RNA成像）实验，并特别着重于对我们的图像处理和数据分析程序的详细描述。
