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Measuring mRNA copy number in individual Escherichia coli cells using single-molecule fluorescent in situ hybridization

Nature Protocols volume 8, pages 1100–1113 (2013)Cite this article

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A Corrigendum to this article was published on 27 August 2015

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Abstract

We present a protocol for measuring the absolute number of mRNA molecules from a gene of interest in individual, chemically fixed Escherichia coli cells. A set of fluorescently labeled oligonucleotide probes is hybridized to the target mRNA, such that each mRNA molecule is decorated by a known number of fluorescent dyes. Cells are then imaged using fluorescence microscopy. The copy number of the target mRNA is estimated from the total intensity of fluorescent foci in the cell, rather than from counting discrete 'spots' as in other currently available protocols. Image analysis is performed using an automated algorithm. The measured mRNA copy number distribution obtained from many individual cells can be used to extract the parameters of stochastic gene activity, namely the frequency and size of transcription bursts from the gene of interest. The experimental procedure takes 2 d, with another 2–3 d typically required for image and data analysis.

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Figure 1: Experimental procedure.
Figure 2: Preparation and use of agarose pads.
Figure 3: Different smFISH samples contain spots of distinct brightness and size.

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Change history

  • 19 August 2015

    In the version of this article initially published, a component (40 µl of 50 mg ml-1 BSA) was erroneously omitted from the 'Hybridization solution' recipe in the Reagent Setup section. The error has been corrected in the HTML and PDF versions of the article.

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Acknowledgements

C. Zong and L.-H. So first introduced the smFISH protocol in our lab. We thank A. Raj, R. Singer and L. Cai for generous advice. We thank all members of the Golding lab for providing help with experiments. The Schnitzcells software was kindly provided by M. Elowitz (California Institute of Technology). Work in the Golding lab was supported by the US National Institutes of Health grant no. R01 GM082837, US National Science Foundation grant nos. 082265 (Physics Frontiers Center: Center for the Physics of Living Cells) and PHY-1147498 (CAREER), Human Frontier Science Program grant no. RGY 70/2008 and Welch Foundation grant no. Q-1759.

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Author notes

  1. Samuel O Skinner and Leonardo A Sepúlveda: These authors contributed equally to this work.

Authors and Affiliations

  1. Verna and Marrs McLean Department of Biochemistry and Molecular Biology, Baylor College of Medicine, Houston, Texas, USA

    Samuel O Skinner, Leonardo A Sepúlveda, Heng Xu & Ido Golding

  2. Department of Physics, University of Illinois, Urbana, Illinois, USA

    Samuel O Skinner & Ido Golding

  3. Center for Biophysics and Computational Biology, University of Illinois, Urbana, Illinois, USA

    Leonardo A Sepúlveda & Ido Golding

  4. Center for the Physics of Living Cells, University of Illinois, Urbana, Illinois, USA

    Heng Xu & Ido Golding

Authors
  1. Samuel O Skinner
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  2. Leonardo A Sepúlveda
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  3. Heng Xu
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  4. Ido Golding
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Contributions

I.G. supervised the project. S.O.S., L.A.S. and H.X. developed the protocol. S.O.S., L.A.S. and I.G. wrote the paper.

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Correspondence to Ido Golding.

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The authors declare no competing financial interests.

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Skinner, S., Sepúlveda, L., Xu, H. et al. Measuring mRNA copy number in individual Escherichia coli cells using single-molecule fluorescent in situ hybridization. Nat Protoc 8, 1100–1113 (2013). https://doi.org/10.1038/nprot.2013.066

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