Detection of the First Round of Translation: The TRICK Assay
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.
Key wordsmRNA translation TRICK MS2/PP7 stem-loops Coat proteins Quantitative fluorescence microscopy in live cells Single-RNA imaging
The authors would like to thank all members of the Chao lab for their joint efforts in developing the TRICK analysis pipeline, in particular, Johannes Wilbertz for providing test data and Ivana Horvathova for generation of the colocalization control. We further thank the Facility for Advanced Imaging and Microscopy at FMI for data acquisition and analysis support. Research in the Chao lab is supported by the Novartis Research Foundation and the Swiss National Science Foundation.
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