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Highly multiplexed simultaneous detection of RNAs and proteins in single cells

Nature Methods volume 13, pages 269275 (2016) | Download Citation

Abstract

To enable the detection of expression signatures specific to individual cells, we developed PLAYR (proximity ligation assay for RNA), a method for highly multiplexed transcript quantification by flow and mass cytometry that is compatible with standard antibody staining. When used with mass cytometry, PLAYR allowed for the simultaneous quantification of more than 40 different mRNAs and proteins. In primary cells, we quantified multiple transcripts, with the identity and functional state of each analyzed cell defined on the basis of the expression of a separate set of transcripts or proteins. By expanding high-throughput deep phenotyping of cells beyond protein epitopes to include RNA expression, PLAYR opens a new avenue for the characterization of cellular metabolism.

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Acknowledgements

We thank L. Lanier (UCSF, San Francisco, California, USA) for providing the NKL cell line, and A. Trejo and A. Jager for technical assistance. A.P.F. is supported by a Fellowship for Prospective Researchers from the Swiss National Science Foundation, an EMBO Long-Term Fellowship and a Marie Curie International Outgoing Fellowship. P.F.G. is a Howard Hughes Medical Institute Fellow of the Life Sciences Research Foundation. F.-A.B. is supported by a Human Frontier Science Program Long-Term Fellowship. This work was supported by the US National Institutes of Health (grants U19 AI057229, 1U19AI100627, R01CA184968, 1R33CA183654-01, R33CA183692, 1R01GM10983601, 1R21CA183660, 1R01NS08953301, OPP1113682, 5UH2AR067676, 1R01CA19665701 and R01HL120724 to G.P.N.), US Department of Defense Congressionally Directed Medical Research Programs (grants OC110674 and 11491122 to G.P.N.), the Northrop-Grumman Corporation (G.P.N.) and the Rachford & Carlotta A. Harris Endowed Chair (G.P.N.).

Author information

Author notes

    • Andreas P Frei
    •  & Felice-Alessio Bava

    These authors contributed equally to this work.

Affiliations

  1. Baxter Laboratory in Stem Cell Biology, Department of Microbiology and Immunology, Stanford University, Stanford, California, USA.

    • Andreas P Frei
    • , Felice-Alessio Bava
    • , Eli R Zunder
    • , Elena W Y Hsieh
    • , Shih-Yu Chen
    • , Garry P Nolan
    •  & Pier Federico Gherardini
  2. Department of Biomedical Engineering, University of Virginia, Charlottesville, Virginia, USA.

    • Eli R Zunder
  3. Department of Pediatrics, University of Colorado Denver, Denver, Colorado, USA.

    • Elena W Y Hsieh
  4. Department of Immunology and Microbiology, University of Colorado Denver, Denver, Colorado, USA.

    • Elena W Y Hsieh

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Contributions

A.P.F., F.-A.B. and P.F.G. conceived the work, performed experiments, analyzed data and wrote the manuscript. E.R.Z. provided help with mouse embryonic stem cell experiments. E.W.Y.H. and S.-Y.C. provided help with cytokine induction experiments. G.P.N. supervised the work and wrote the manuscript.

Competing interests

G.P.N. has a personal financial interest in the company Fluidigm, the manufacturer of the mass cytometer used in this study.

Corresponding authors

Correspondence to Garry P Nolan or Pier Federico Gherardini.

Integrated supplementary information

Supplementary information

PDF files

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    Supplementary Text and Figures

    Supplementary Figures 1–7

Excel files

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    Supplementary Table 1

    PLAYR probes and backbone-insert systems

Zip files

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    Supplementary Software

    PLAYR Design software for probe design

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DOI

https://doi.org/10.1038/nmeth.3742

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