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Curr Protoc Mol Biol. 2016;113:14.24.1-14.24.15. Epub 2016 Jan 4.

Single-Molecule mRNA Detection in Live Yeast.

Author information

1
Center for Cancer Research, National Cancer Institute, Building 41, Room B302, 41 Library Dr., Bethesda, MD 20814, Tineke.Lenstra@nih.gov , 301-496-4793.
2
Center for Cancer Research, National Cancer Institute, Building 41, Room B201, 41 Library Dr., Bethesda, MD 20814, Dan.Larson@nih.gov , 301-496-0986.

Abstract

Visualization of single RNA molecules in living cells has enabled the study of synthesis, movement, and localization of mRNAs and has provided insight into gene regulation with sub-second temporal resolution and nanometer spatial resolution. Following transcription in single cells indicates that gene activity is heterogeneous between cells and also exhibits random variability over time even within single cells. Studies of mRNAs in yeast can take advantage of the powerful genetics available in this model organism and allow mechanistic questions to be addressed. In this chapter, we describe an approach for visualizing mRNA and transcription in live yeast cells. The method is based on binding of fluorescently labeled MS2 and PP7 coat proteins to stem loops sequences that are introduced into the gene of interest. We give detailed protocols for the construction of the necessary yeast strains, for image acquisition, and for validation.

KEYWORDS:

RNA; Single-molecule; fluorescence; live cell; microscopy; transcription

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