Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

  • Protocol
  • Published:

FISH-Flow, a protocol for the concurrent detection of mRNA and protein in single cells using fluorescence in situ hybridization and flow cytometry

Nature Protocols volume 12, pages 1245–1260 (2017)Cite this article

Subjects

Abstract

We describe a flow-cytometry-based protocol for intracellular mRNA measurements in nonadherent mammalian cells using fluorescence in situ hybridization (FISH) probes. The method, which we call FISH-Flow, allows for high-throughput multiparametric measurements of gene expression, a task that was not feasible with earlier, microscopy-based approaches. The FISH-Flow protocol involves cell fixation, permeabilization and hybridization with a set of fluorescently labeled oligonucleotide probes. In this protocol, surface and intracellular protein markers can also be stained with fluorescently labeled antibodies for simultaneous protein and mRNA measurement. Moreover, a semiautomated, single-tube version of the protocol can be performed with a commercially available cell-wash device that reduces cell loss, operator time and interoperator variability. It takes ∼30 h to perform this protocol. An example of FISH-Flow measurements of cytokine mRNA induction by ex vivo stimulation of primed T cells with specific antigens is described.

This is a preview of subscription content, access via your institution

Access options

Buy this article

  • Purchase on Springer Link
  • Instant access to full article PDF
Buy now

Prices may be subject to local taxes which are calculated during checkout

Figure 1: Comparison of mRNA expression analysis by microscopy and FISH-Flow.
Figure 2: Analysis of IFNG mRNA expression in T cells using manual and LWA semiautomated FISH-Flow protocols.
Figure 3: Flow cytometric analysis of Irga6 mRNA and IRGA6 protein in mouse macrophage cell line RAW264.7.
Figure 4: Installation of the external tank in the LWA instrument.
Figure 5: Multiplex five-color analysis of IFNG mRNA expression in PBMC subsets.

Similar content being viewed by others

References

  1. Sanchez, A. & Golding, I. Genetic determinants and cellular constraints in noisy gene expression. Science 342, 1188–1193 (2013).

    Article  CAS  Google Scholar 

  2. 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 

  3. 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 

  4. Rahman, S. & Zenklusen, D. Single-molecule resolution fluorescent in situ hybridization (smFISH) in the yeast S. cerevisiae. Methods Mol. Biol. 1042, 33–46 (2013).

    Article  CAS  Google Scholar 

  5. Batish, M., Raj, A. & Tyagi, S. Single molecule imaging of RNA in situ. Methods Mol. Biol. 714, 3–13 (2011).

    Article  CAS  Google Scholar 

  6. Tyagi, S. Imaging intracellular RNA distribution and dynamics in living cells. Nat. Methods 6, 331–338 (2009).

    Article  CAS  Google Scholar 

  7. Bushkin, Y. et al. Profiling T cell activation using single-molecule fluorescence in situ hybridization and flow cytometry. J. Immunol. 194, 836–841 (2015).

    Article  CAS  Google Scholar 

  8. Vir, P. et al. Single-cell cytokine gene expression in peripheral blood cells correlates with latent tuberculosis status. PLoS One 10, e0144904 (2015).

    Article  Google Scholar 

  9. Chattopadhyay, P.K. & Roederer, M. Cytometry: today's technology and tomorrow's horizons. Methods 57, 251–258 (2012).

    Article  CAS  Google Scholar 

  10. Hanley, M.B., Lomas, W., Mittar, D., Maino, V. & Park, E. Detection of low abundance RNA molecules in individual cells by flow cytometry. PLoS One 8, e57002 (2013).

    Article  CAS  Google Scholar 

  11. Porichis, F. et al. Differential impact of PD-1 and/or interleukin-10 blockade on HIV-1-specific CD4 T cell and antigen-presenting cell functions. J. Virol. 88, 2508–2518 (2014).

    Article  Google Scholar 

  12. Larsson, H.M. et al. Sorting live stem cells based on Sox2 mRNA expression. PLoS One 7, e49874 (2012).

    Article  CAS  Google Scholar 

  13. McClellan, S. et al. mRNA detection in living cells: A next generation cancer stem cell identification technique. Methods 82, 47–54 (2015).

    Article  CAS  Google Scholar 

  14. Perfetto, S.P., Chattopadhyay, P.K. & Roederer, M. Seventeen-colour flow cytometry: unravelling the immune system. Nat. Rev. Immunol. 4, 648–655 (2004).

    Article  CAS  Google Scholar 

  15. Hulspas, R., O'Gorman, M.R., Wood, B.L., Gratama, J.W. & Sutherland, D.R. Considerations for the control of background fluorescence in clinical flow cytometry. Cytometry B Clin. Cytom. 76, 355–364 (2009).

    Article  Google Scholar 

  16. Perfetto, S.P. et al. Amine-reactive dyes for dead cell discrimination in fixed samples. Curr. Protoc. Cytom. Chapter 9, Unit 9.34, (2010).

  17. Perfetto, S.P., Ambrozak, D., Nguyen, R., Chattopadhyay, P.K. & Roederer, M. Quality assurance for polychromatic flow cytometry using a suite of calibration beads. Nat. Protoc. 7, 2067–2079 (2012).

    Article  CAS  Google Scholar 

  18. Roederer, M. How many events is enough? Are you positive? Cytometry A 73, 384–385 (2008).

    Article  Google Scholar 

  19. 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 

  20. Shah, K. & Tyagi, S. Barriers to transmission of transcriptional noise in a c-fos c-jun pathway. Mol. Syst. Biol. 9, 687 (2013).

    Article  CAS  Google Scholar 

  21. Caligiuri, M.A. Human natural killer cells. Blood 112, 461–469 (2008).

    Article  CAS  Google Scholar 

  22. Sester, U. et al. Whole-blood flow-cytometric analysis of antigen-specific CD4 T-cell cytokine profiles distinguishes active tuberculosis from non-active states. PLoS One 6, e17813 (2011).

    Article  CAS  Google Scholar 

Download references

Acknowledgements

This work was supported by grants from the National Institutes of Health (AI-104615, AI-106036 and AI-124691) and by an intramural grant from the Rutgers Office of Research and Economic Development/New Jersey Health Foundation. We are indebted to S.A.E. Marras of the Public Health Research Institute and S. Singh of the Rutgers NJMS Flow Cytometry and Immunology Core Laboratory for constant assistance, advice and superb technical support. We are also grateful to K. Drlica for critical comments on the manuscript.

Author information

Authors and Affiliations

  1. Public Health Research Institute, New Jersey Medical School, Rutgers, The State University of New Jersey, Newark, New Jersey, USA

    Riccardo Arrigucci, Yuri Bushkin, Felix Radford, Karim Lakehal, Pooja Vir, Richard Pine, Sanjay Tyagi & Maria Laura Gennaro

  2. BD Biosciences, San Jose, California, USA

    December Martin & Jeffrey Sugarman

  3. Center for Immunity and Inflammation, New Jersey Medical School, Rutgers, The State University of New Jersey, Newark, New Jersey, USA

    Yanlin Zhao & George S Yap

  4. Global Tuberculosis Institute, New Jersey Medical School, Rutgers, The State University of New Jersey, Newark, New Jersey, USA

    Alfred A Lardizabal

Authors
  1. Riccardo Arrigucci
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Yuri Bushkin
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Felix Radford
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Karim Lakehal
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Pooja Vir
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Richard Pine
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. December Martin
    View author publications

    You can also search for this author in PubMed Google Scholar

  8. Jeffrey Sugarman
    View author publications

    You can also search for this author in PubMed Google Scholar

  9. Yanlin Zhao
    View author publications

    You can also search for this author in PubMed Google Scholar

  10. George S Yap
    View author publications

    You can also search for this author in PubMed Google Scholar

  11. Alfred A Lardizabal
    View author publications

    You can also search for this author in PubMed Google Scholar

  12. Sanjay Tyagi
    View author publications

    You can also search for this author in PubMed Google Scholar

  13. Maria Laura Gennaro
    View author publications

    You can also search for this author in PubMed Google Scholar

Contributions

R.A., M.L.G. and S.T. conceived and designed the experiments. R.A. and S.T. performed the experiments. R.A., M.L.G. and S.T. analyzed the data. Y.B. and R.P. contributed to the initial FISH-Flow protocol. F.R., K.L. and P.V. contributed to the initial FISH-Flow experiments. D.M. and J.S. developed the modified LWA instrument and contributed to semiautomated protocol design and data analysis. Y.Z. and G.S.Y. designed and provided cells for the concurrent FISH-Flow/ICS time-course experiments. S.T. developed the RNA FISH probes. A.A.L. obtained blood samples from patients. M.L.G. supervised the study. R.A., M.L.G. and S.T. wrote the manuscript.

Corresponding author

Correspondence to Sanjay Tyagi.

Ethics declarations

Competing interests

Rutgers University receives royalties from the sale of prelabeled sm-FISH probes by Biosearch Technologies, which markets them as Stellaris probes. A fraction of these proceeds is distributed to S.T.'s laboratory for research and to him personally. D.M. and J.S. are currently employed by BD Biosciences. Additional patent applications related to this technology coauthored by Y.B., M.L.G., S.T. and R.P. are pending. These proceeds, affiliations and patent applications do not influence the conclusions of this research.

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Arrigucci, R., Bushkin, Y., Radford, F. et al. FISH-Flow, a protocol for the concurrent detection of mRNA and protein in single cells using fluorescence in situ hybridization and flow cytometry. Nat Protoc 12, 1245–1260 (2017). https://doi.org/10.1038/nprot.2017.039

Download citation

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/nprot.2017.039

This article is cited by

Comments

By submitting a comment you agree to abide by our Terms and Community Guidelines. If you find something abusive or that does not comply with our terms or guidelines please flag it as inappropriate.

Search

Advanced search

Quick links

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing