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      Imaging individual mRNA molecules using multiple singly labeled probes

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          Abstract

          We describe a method for imaging individual mRNA molecules in fixed cells by probing each mRNA species with 48 or more short, singly labeled oligonucleotide probes. This makes each mRNA molecule visible as a computationally identifiable fluorescent spot via fluorescence microscopy. We demonstrate simultaneous detection of three mRNA species in single cells and mRNA detection in yeast, nematodes, fruit fly wing discs, mammalian cell lines and neurons.

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          Most cited references14

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          Visualization of single RNA transcripts in situ.

          Fluorescence in situ hybridization (FISH) and digital imaging microscopy were modified to allow detection of single RNA molecules. Oligodeoxynucleotide probes were synthesized with five fluorochromes per molecule, and the light emitted by a single probe was calibrated. Points of light in exhaustively deconvolved images of hybridized cells gave fluorescent intensities and distances between probes consistent with single messenger RNA molecules. Analysis of beta-actin transcription sites after serum induction revealed synchronous and cyclical transcription from single genes. The rates of transcription initiation and termination and messenger RNA processing could be determined by positioning probes along the transcription unit. This approach extends the power of FISH to yield quantitative molecular information on a single cell.
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            Noise in gene expression determines cell fate in Bacillus subtilis.

            Random cell-to-cell variations in gene expression within an isogenic population can lead to transitions between alternative states of gene expression. Little is known about how these variations (noise) in natural systems affect such transitions. In Bacillus subtilis, noise in ComK, the protein that regulates competence for DNA uptake, is thought to cause cells to transition to the competent state in which genes encoding DNA uptake proteins are expressed. We demonstrate that noise in comK expression selects cells for competence and that experimental reduction of this noise decreases the number of competent cells. We also show that transitions are limited temporally by a reduction in comK transcription. These results illustrate how such stochastic transitions are regulated in a natural system and suggest that noise characteristics are subject to evolutionary forces.
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              Fluorescence in situ hybridization: past, present and future.

              Fluorescence in situ hybridization (FISH), the assay of choice for localization of specific nucleic acids sequences in native context, is a 20-year-old technology that has developed continuously. Over its maturation, various methodologies and modifications have been introduced to optimize the detection of DNA and RNA. The pervasiveness of this technique is largely because of its wide variety of applications and the relative ease of implementation and performance of in situ studies. Although the basic principles of FISH have remained unchanged, high-sensitivity detection, simultaneous assay of multiple species, and automated data collection and analysis have advanced the field significantly. The introduction of FISH surpassed previously available technology to become a foremost biological assay. Key methodological advances have allowed facile preparation of low-noise hybridization probes, and technological breakthroughs now permit multi-target visualization and quantitative analysis - both factors that have made FISH accessible to all and applicable to any investigation of nucleic acids. In the future, this technique is likely to have significant further impact on live-cell imaging and on medical diagnostics.
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                Author and article information

                Journal
                101215604
                32338
                Nat Methods
                Nat. Methods
                Nature methods
                1548-7091
                1548-7105
                8 June 2011
                21 September 2008
                October 2008
                29 June 2011
                : 5
                : 10
                : 877-879
                Affiliations
                [1 ] Department of Physics, Massachusetts Institute of Technology, 77 Massachusetts Avenue, Cambridge, MA 02139
                [2 ] Public Health Research Institute Center, New Jersey Medical School-University of Medicine and Dentistry of New Jersey, 225 Warren Street, Newark, NJ 07103
                Author notes
                Article
                nihpa67303
                10.1038/nmeth.1253
                3126653
                18806792
                7346828a-b806-4e39-a6e3-df5612892cb0
                History
                Funding
                Funded by: National Institute of General Medical Sciences : NIGMS
                Award ID: R01 GM077183-04 || GM
                Funded by: National Institute of General Medical Sciences : NIGMS
                Award ID: R01 GM077183-03 || GM
                Funded by: National Institute of General Medical Sciences : NIGMS
                Award ID: R01 GM068957-07 || GM
                Funded by: National Institute of General Medical Sciences : NIGMS
                Award ID: R01 GM068957-06 || GM
                Categories
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                Life sciences
                Life sciences

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