RNA Detection pp 373-384 | Cite as

Detection of the First Round of Translation: The TRICK Assay

Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 1649)

Abstract

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 words

mRNA translation TRICK MS2/PP7 stem-loops Coat proteins Quantitative fluorescence microscopy in live cells Single-RNA imaging 

Notes

Acknowledgments

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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Copyright information

© Springer Science+Business Media LLC 2018

Authors and Affiliations

  1. 1.Friedrich Miescher Institute for Biomedical ResearchBaselSwitzerland

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