Guet, Calin C.Bruneaux, Luke JulienMin, Taejin L.Siegal-Gaskins, DanFigueroa, IsraelEmonet, ThierryCluzel, Philippe2009-08-252008Guet, Calin C., Luke Bruneaux, Taejin L. Min, Dan Siegal-Gaskins, Israel Figueroa, Thierry Emonet, and Philippe Cluzel. 2008. Minimally invasive determination of mRNA concentration in single living bacteria. Nucleic Acids Research 36(12): e73.0305-10480301-5610http://nrs.harvard.edu/urn-3:HUL.InstRepos:3224722Fluorescence 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 »40nM (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.en-USJournal Article10.1093/nar/gkn329