Protocol | Published:

Multiplexed profiling of RNA and protein expression signatures in individual cells using flow or mass cytometry

Nature Protocols (2019) | Download Citation


Advances in single-cell analysis technologies are providing novel insights into phenotypic and functional heterogeneity within seemingly identical cell populations. RNA within single cells can be analyzed using unbiased sequencing protocols or through more targeted approaches using in situ hybridization (ISH). The proximity ligation assay for RNA (PLAYR) approach is a sensitive and high-throughput technique that relies on in situ and proximal ligation to measure at least 27 specific RNAs by flow or mass cytometry. We provide detailed instructions for combining this technique with antibody-based detection of surface/internal protein, allowing simultaneous highly multiplexed profiling of RNA and protein expression at single-cell resolution. PLAYR overcomes limitations on multiplexing seen in previous branching DNA–based RNA detection techniques by integration of a transcript-specific oligonucleotide sequence within a rolling-circle amplification (RCA). This unique transcript-associated sequence can then be detected by heavy metal (for mass cytometry)- or fluorophore (for flow cytometry)-conjugated complementary detection oligonucleotides. Included in this protocol is methodology to label oligonucleotides with lanthanide metals for use in mass cytometry. When analyzed by mass cytometry, up to 40 variables (with scope for future expansion) can be measured simultaneously. We used the described protocol to demonstrate intraclonal heterogeneity within primary cells from chronic lymphocytic leukemia patients, but it can be adapted to other primary cells or cell lines in suspension. This robust, reliable and reproducible protocol can be completed in 2–3 d and can be paused at several stages for convenience.

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The data that support the findings of this study are available from the authors on reasonable request.

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The work described was supported by grants provided by Northwest Cancer Research, UK (NWCR138752); The Bloom Appeal; and The Isle of Man Cancer Fund (A.D.D., J.R.S. and N.K.).

Author information

Author notes

  1. These authors contributed equally: A. D. Duckworth, P. F. Gherardini.

  2. These authors jointly supervised this work: J. R. Slupsky, N. Kalakonda.


  1. Department of Molecular and Clinical Cancer Medicine, Institute of Translational Medicine, University of Liverpool, Liverpool, UK

    • Andrew D. Duckworth
    • , Martina Sykorova
    • , Faten Yasin
    • , Joseph R. Slupsky
    •  & Nagesh Kalakonda
  2. Baxter Laboratory in Stem Cell Biology, Department of Microbiology and Immunology, Stanford University, Stanford, CA, USA

    • Pier Federico Gherardini
    •  & Garry P. Nolan
  3. Parker Institute for Cancer Immunotherapy, San Francisco, CA, USA

    • Pier Federico Gherardini


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A.D.D., M.S. and F.Y. performed the experiments. A.D.D. and P.F.G. collaborated on assay development and optimization. A.D.D., P.F.G., G.P.N., J.R.S. and N.K. contributed to critical analysis of data and manuscript preparation.

Competing interests

G.P.N. had a personal financial interest in Fluidigm, the manufacturer of the mass cytometer used in this study, for the duration of this project. The other authors declare no competing interests.

Corresponding author

Correspondence to Nagesh Kalakonda.

Integrated supplementary information

  1. Supplementary Figure 1 Performance of PLAYR assay with and without the methanol step.

    Flow cytometry analysis of GAPDH mRNA expression in the MAVER1 cell line. Assay was performed with or without the methanol (MeOH) step from the PLAYR assay. Signal is compared to a negative control in which the rolling circle amplification step was performed without addition of Phi29 polymerase. ATTO647 is an Alexa647 equivalent available from IDT technologies.

  2. Supplementary Figure 2 Detection of RNA using less stringent PLAYR conditions.

    . Mass cytometric analysis of MAVER1 cells treated with 100nM PMA and 1µM Ionomycin for the times indicated. After treatment, cells at each time point were split and either analyzed for RNAs (1-7 and ACTB; multiplexed) using the original PLAYR protocol or using optimized but milder conditions (37oC Probe hybridization/post hybridization wash, originally 40oC; 0.2% Tween20 in hybridization buffer, originally 1% Tween20; and 2X saline–sodium citrate solution used in post hybridization wash, originally 4X saline–sodium citrate solution). Upon completion of probe hybridization and post hybridization wash steps, all samples were pooled together using palladium barcoding for downstream steps in the assay.

Supplementary information

  1. Supplementary Text and Figures

    Supplementary Figures 1 and 2

  2. Reporting Summary

  3. Supplementary Table 1

    Supplementary Table 1

  4. Supplementary Table 2

    Supplementary Table 2

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