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Nucleic Acids Research Advance Access published online on May 30, 2008
Nucleic Acids Research, doi:10.1093/nar/gkn329
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© 2008 The Author(s)
This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/2.0/uk/) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.

Methods Online

Minimally invasive determination of mRNA concentration in single living bacteria

Calin C. Guet, Luke Bruneaux, Taejin L. Min, Dan Siegal-Gaskins, Israel Figueroa, Thierry Emonet and Philippe Cluzel*

The James Franck Institute, Institute for Biophysical Dynamics, Department of Physics, The University of Chicago, 929 E 57th St, Chicago, IL 60637, USA

*To whom correspondence should be addressed. Tel: 773-834-9096; Email: cluzel{at}uchicago.edu

Received January 18, 2008. Revised May 7, 2008. Accepted May 8, 2008.

Fluorescence correlation spectroscopy (FCS) has permitted the characterization of high concentrations of noncoding RNAs in a single living bacterium. Here, we extend the use of FCS to low concentrations of coding RNAs in single living cells. We genetically fuse a red fluorescent protein (RFP) gene and two binding sites for an RNA-binding protein, whose translated product is the RFP protein alone. Using this construct, we determine in single cells both the absolute [mRNA] concentration and the associated [RFP] expressed from an inducible plasmid. We find that the FCS method allows us to reliably monitor in real-time [mRNA] down to ~40 nM (i.e. approximately two transcripts per volume of detection). To validate these measurements, we show that [mRNA] is proportional to the associated expression of the RFP protein. This FCS-based technique establishes a framework for minimally invasive measurements of mRNA concentration in individual living bacteria.

Present addresses: Taejin L. Min, Department of Physics, 1110 West Green, University of Illinois at Urbana Champaign Urbana, IL 61801, USA

Thierry Emonet, MCDB, Yale University New Haven, CT 06510, USA


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