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Methods. 2019 Jun 1;162-163:12-22. doi: 10.1016/j.ymeth.2019.03.021. Epub 2019 Mar 21.

Translation imaging of single mRNAs in established cell lines and primary cultured neurons.

Author information

1
Johns Hopkins School of Medicine, Department of Biophysics and Biophysical Chemistry, 855 N Wolfe Street Ste. 454, Baltimore, MD 21205, USA; Johns Hopkins School of Medicine, Center for Cell Dynamics, Baltimore, USA.
2
Johns Hopkins School of Medicine, Department of Biophysics and Biophysical Chemistry, 855 N Wolfe Street Ste. 454, Baltimore, MD 21205, USA; Johns Hopkins School of Medicine, Center for Cell Dynamics, Baltimore, USA; Johns Hopkins School of Medicine, Solomon H. Snyder Department of Neuroscience, Baltimore, USA. Electronic address: bwu20@jhmi.edu.

Abstract

The central dogma of molecular biology reaches a crescendo at its final step: the translation of an mRNA into its corresponding protein product. This process is highly regulated both spatially and temporally, requiring techniques to interrogate the subcellular translational status of mRNAs in both living and fixed cells. Single-molecule imaging of nascent peptides (SINAPs) and related techniques allow us to study this fundamental process for single mRNAs in live cells. These techniques enable researchers to address previously intractable questions in the central dogma, such as the origin of stochastic translational control and the role of local translation in highly polarized cells. In this review, we present the methodology and the theoretical framework for conducting studies using SINAPs in both established cell lines and primary cultured neurons.

KEYWORDS:

Fluorescence microscopy; Single-molecule; Translation; mRNA

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