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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.

References

  1. Femino, A.M., Fay, F.S., Fogarty, K. & Singer, R.H. Visualization of single RNA transcripts in situ. Science 280, 585–590 (1998).

    Article  CAS  Google Scholar 

  2. Raj, A., Peskin, C.S., Tranchina, D., Vargas, D.Y. & Tyagi, S. Stochastic mRNA synthesis in mammalian cells. PLoS Biol. 4, e309 (2006).

    Article  Google Scholar 

  3. Zenklusen, D., Larson, D.R. & Singer, R.H. Single-RNA counting reveals alternative modes of gene expression in yeast. Nat. Struct. Mol. Biol. 15, 1263–1271 (2008).

    Article  CAS  Google Scholar 

  4. Raj, A., van den Bogaard, P., Rifkin, S.A., van Oudenaarden, A. & Tyagi, S. Imaging individual mRNA molecules using multiple singly labeled probes. Nat. Methods 5, 877–879 (2008).

    Article  CAS  Google Scholar 

  5. Taniguchi, Y. et al. Quantifying E. coli proteome and transcriptome with single-molecule sensitivity in single cells. Science 329, 533–538 (2010).

    Article  CAS  Google Scholar 

  6. Trcek, T. et al. Single-mRNA counting using fluorescent in situ hybridization in budding yeast. Nat. Protoc. 7, 408–419 (2012).

    Article  CAS  Google Scholar 

  7. Zenklusen, D. & Singer, R.H. Analyzing mRNA expression using single mRNA resolution fluorescent in situ hybridization. Methods Enzymol. 470, 641–659 (2010).

    Article  CAS  Google Scholar 

  8. Zong, C., So, L.H., Sepulveda, L.A., Skinner, S.O. & Golding, I. Lysogen stability is determined by the frequency of activity bursts from the fate-determining gene. Mol. Syst. Biol. 6, 440 (2010).

    Article  Google Scholar 

  9. So, L.H. et al. General properties of transcriptional time series in Escherichia coli. Nat. Genet. 43, 554–560 (2011).

    Article  CAS  Google Scholar 

  10. Golding, I., Paulsson, J., Zawilski, S.M. & Cox, E.C. Real-time kinetics of gene activity in individual bacteria. Cell 123, 1025–1036 (2005).

    Article  CAS  Google Scholar 

  11. Thompson, R.E., Larson, D.R. & Webb, W.W. Precise nanometer localization analysis for individual fluorescent probes. Biophys. J. 82, 2775–2283 (2002).

    Article  CAS  Google Scholar 

  12. Lubeck, E. & Cai, L. Single-cell systems biology by super-resolution imaging and combinatorial labeling. Nat. Methods 9, 743–748 (2012).

    Article  CAS  Google Scholar 

  13. Golding, I. & Cox, E.C. Chapter 8: Spatiotemporal dynamics in bacterial cells: real-time studies with single-event resolution. Methods Cell Biol. 89, 223–251 (2008).

    Article  CAS  Google Scholar 

  14. Maamar, H., Raj, A. & Dubnau, D. Noise in gene expression determines cell fate in Bacillus subtilis. Science 317, 526–529 (2007).

    Article  CAS  Google Scholar 

  15. Kafri, R. et al. Dynamics extracted from fixed cells reveal feedback linking cell growth to cell cycle. Nature 494, 480–483 (2013).

    Article  CAS  Google Scholar 

  16. Peccoud, J. & Ycart, B. Markovian modeling of gene-product synthesis. Theor. Popul. Biol. 48, 222–234 (1995).

    Article  Google Scholar 

  17. Shahrezaei, V. & Swain, P.S. Analytical distributions for stochastic gene expression. Proc. Natl. Acad. Sci. USA 105, 17256–17261 (2008).

    Article  CAS  Google Scholar 

  18. Levsky, J.M., Shenoy, S.M., Pezo, R.C. & Singer, R.H. Single-cell gene expression profiling. Science 297, 836–840 (2002).

    Article  CAS  Google Scholar 

  19. Long, R.M. et al. Mating type switching in yeast controlled by asymmetric localization of ASH1 mRNA. Science 277, 383–387 (1997).

    Article  CAS  Google Scholar 

  20. Golding, I. & Cox, E.C. RNA dynamics in live Escherichia coli cells. Proc. Natl. Acad. Sci. USA 101, 11310–11315 (2004).

    Article  CAS  Google Scholar 

  21. Montero Llopis, P. et al. Spatial organization of the flow of genetic information in bacteria. Nature 466, 77–81 (2010).

    Article  Google Scholar 

  22. Nevo-Dinur, K., Nussbaum-Shochat, A., Ben-Yehuda, S. & Amster-Choder, O. Translation-independent localization of mRNA in E. coli. Science 331, 1081–1084 (2011).

    Article  CAS  Google Scholar 

  23. Bakshi, S., Siryaporn, A., Goulian, M. & Weisshaar, J.C. Superresolution imaging of ribosomes and RNA polymerase in live Escherichia coli cells. Mol. Microbiol. 85, 21–38 (2012).

    Article  CAS  Google Scholar 

  24. Kuhlman, T.E. & Cox, E.C. Gene location and DNA density determine transcription factor distributions in Escherichia coli. Mol. Syst. Biol. 8, 610 (2012).

    Article  Google Scholar 

  25. Phillips, R., Kondev, J., Theriot, J. & Garcia, H. Physical Biology of the Cell (Garland Science, 2012).

  26. Yu, J., Xiao, J., Ren, X., Lao, K. & Xie, X.S. Probing gene expression in live cells, one protein molecule at a time. Science 311, 1600–1603 (2006).

    Article  CAS  Google Scholar 

  27. Hebenstreit, D. et al. RNA sequencing reveals two major classes of gene expression levels in metazoan cells. Mol. Syst. Biol. 7, 497 (2011).

    Article  Google Scholar 

  28. Bertrand, E. et al. Localization of ASH1 mRNA particles in living yeast. Mol. Cell 2, 437–445 (1998).

    Article  CAS  Google Scholar 

  29. Fusco, D. et al. Single mRNA molecules demonstrate probabilistic movement in living mammalian cells. Curr. Biol. 13, 161–167 (2003).

    Article  CAS  Google Scholar 

  30. Young, J.W. et al. Measuring single-cell gene expression dynamics in bacteria using fluorescence time-lapse microscopy. Nat. Protoc. 7, 80–88 (2012).

    Article  CAS  Google Scholar 

  31. Cohen, A.A. et al. Dynamic proteomics of individual cancer cells in response to a drug. Science 322, 1511–1516 (2008).

    Article  CAS  Google Scholar 

  32. Geva-Zatorsky, N. et al. Protein dynamics in drug combinations: a linear superposition of individual-drug responses. Cell 140, 643–651 (2010).

    Article  CAS  Google Scholar 

  33. Neidhardt, F.C. (ed.) Escherichia coli and Salmonella: Cellular and Molecular Biology (ASM Press, 1996).

  34. Bates, D. & Kleckner, N. Chromosome and replisome dynamics in E. coli: loss of sister cohesion triggers global chromosome movement and mediates chromosome segregation. Cell 121, 899–911 (2005).

    Article  CAS  Google Scholar 

  35. Kuhlman, T., Zhang, Z., Saier, M.H. Jr. & Hwa, T. Combinatorial transcriptional control of the lactose operon of Escherichia coli. Proc. Natl. Acad. Sci. USA 104, 6043–6048 (2007).

    Article  CAS  Google Scholar 

  36. Yakovleva, G.M., Kim, S.K. & Wanner, B.L. Phosphate-independent expression of the carbon-phosphorus lyase activity of Escherichia coli. Appl. Microbiol. Biotechnol. 49, 573–588 (1998).

    Article  CAS  Google Scholar 

  37. Raj, A. & Tyagi, S. Detection of individual endogenous RNA transcripts in situ using multiple singly labeled probes. Methods Enzymol. 472, 365–386 (2010).

    Article  CAS  Google Scholar 

  38. Batish, M., Raj, A. & Tyagi, S. Single molecule imaging of RNA in situ. Methods Mol. Biol. 714, 3–13 (2010).

    Article  Google Scholar 

  39. Sliusarenko, O., Heinritz, J., Emonet, T. & Jacobs-Wagner, C. High-throughput, subpixel precision analysis of bacterial morphogenesis and intracellular spatio-temporal dynamics. Mol. Microbiol. 80, 612–627 (2011).

    Article  CAS  Google Scholar 

  40. Paulsson, J. & Ehrenberg, M. Random signal fluctuations can reduce random fluctuations in regulated components of chemical regulatory networks. Phys. Rev. Lett. 84, 5447–5450 (2000).

    Article  CAS  Google Scholar 

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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.

Corresponding author

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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