title: Estimation of fluorescence-tagged RNA numbers from spot intensities.
abstract: Present research on gene expression using live cell imaging and fluorescent proteins or tagged RNA requires accurate automated methods of quantification of these molecules from the images. Here, we propose a novel automated method for classifying pixel intensities of fluorescent spots to RNA numbers.
  
  
  
  The method relies on a new model of intensity distributions of tagged RNAs, for which we estimated parameter values in maximum likelihood sense from measurement data, and constructed a maximum a posteriori classifier to estimate RNA numbers in fluorescent RNA spots. We applied the method to estimate the number of tagged RNAs in individual live Escherichia coli cells containing a gene coding for an RNA with MS2-GFP binding sites. We tested the method using two constructs, coding for either 96 or 48 binding sites, and obtained similar distributions of RNA numbers, showing that the method is adaptive. We further show that the results agree with a method that uses time series data and with quantitative polymerase chain reaction measurements. Lastly, using simulated data, we show that the method is accurate in realistic parameter ranges. This method should, in general, be applicable to live single-cell measurements of low-copy number fluorescence-tagged molecules.
  
  
  
  MATLAB extensions written in C for parameter estimation and finding decision boundaries are available under Mozilla public license at http://www.cs.tut.fi/%7ehakkin22/estrna/ CONTACT: andre.ribeiro@tut.fi.
authors.affiliation: Department of Signal Processing, Laboratory of Biosystem Dynamics, Computational Systems Biology Research Group, Tampere University of Technology, Tampere, Finland.
authors.forename: Antti
authors.initials: A
authors.lastname: Häkkinen
authors.affiliation: Department of Signal Processing, Laboratory of Biosystem Dynamics, Computational Systems Biology Research Group, Tampere University of Technology, Tampere, Finland.
authors.forename: Meenakshisundaram
authors.initials: M
authors.lastname: Kandhavelu
authors.affiliation: Department of Signal Processing, Laboratory of Biosystem Dynamics, Computational Systems Biology Research Group, Tampere University of Technology, Tampere, Finland.
authors.forename: Stefania
authors.initials: S
authors.lastname: Garasto
authors.affiliation: Department of Signal Processing, Laboratory of Biosystem Dynamics, Computational Systems Biology Research Group, Tampere University of Technology, Tampere, Finland.
authors.forename: Andre S
authors.initials: AS
authors.lastname: Ribeiro
mesh_terms: D004926:Escherichia coli / Q000235:genetics; D005453:Fluorescence; D008856:Microscopy, Fluorescence; D012329:RNA, Bacterial / Q000737:chemistry*
chemical_list: D012329:RNA, Bacterial