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Methods Enzymol. 2010;470:641-59. doi: 10.1016/S0076-6879(10)70026-4. Epub 2010 Mar 1.

Analyzing mRNA expression using single mRNA resolution fluorescent in situ hybridization.

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  • 1Department of Anatomy and Structural Biology and The Gruss-Lipper Biophotonics Center, Albert Einstein College of Medicine, Bronx, New York, USA.

Abstract

As the product of transcription and the blueprint for translation, mRNA is the main intermediate product of the gene expression pathway. The ability to accurately determine mRNA levels is, therefore, a major requirement when studying gene expression. mRNA is also a target of different regulatory steps, occurring in different subcellular compartments. To understand the different steps of gene expression regulation, it is therefore essential to analyze mRNA in the context of a single cell, maintaining spatial information. Here, we describe a stepwise protocol for fluorescent in situ hybridization (FISH) that allows detection of individual mRNAs in single yeast cells. This method allows quantitative analysis of mRNA expression in single cells, permitting "absolute" quantification by simply counting mRNAs. It further allows us to study many aspects of mRNA metabolism, from transcription to processing, localization, and mRNA degradation.

Copyright © 2010 Elsevier Inc. All rights reserved.

PMID:
20946829
[PubMed - indexed for MEDLINE]
PMCID:
PMC3162037
Free PMC Article
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