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Highly multiplexed simultaneous detection of RNAs and proteins in single cells

Nature Methods volume 13, pages 269–275 (2016)Cite this article

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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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Figure 1: PLAYR enables the simultaneous quantification of specific transcripts and proteins in single cells.
Figure 2: Highly multiplexed measurement of different transcripts in single cells.
Figure 3: Highly multiplexed measurement of transcripts in cell types defined by other transcripts or protein epitopes.
Figure 4: Measurements of cytokine transcript induction in human PBMCs by mass cytometry and fluorescence flow cytometry.
Figure 5: Single-cell-resolution map of cytokine induction in human PBMCs.

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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.).

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Author notes

  1. Andreas P Frei and Felice-Alessio Bava: These authors contributed equally to this work.

Authors and Affiliations

  1. Department of Microbiology and Immunology, Baxter Laboratory in Stem Cell Biology, 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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  1. Andreas P Frei
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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.

Corresponding authors

Correspondence to Garry P Nolan or Pier Federico Gherardini.

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Competing interests

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

Integrated supplementary information

Supplementary Figure 1 Validation of different PLAYR insert systems used for multiplexed detection of transcripts.

The three transcripts ACTB, GAPDH, and PPIA were detected using 2 probe pairs per gene on 7 different insert systems used for multiplexed detection of transcripts. Rolling Circle Amplification was carried out for 30min, 1h, 2h, 4h or over night (16h) and samples were analyzed by mass cytometry.

Source data

Supplementary Figure 2 Graphical display of the PLAYRDesign software tool for user-friendly design of PLAYR probe pairs.

Each potential probe is represented by a red rectangle. The Primer3 score of each probe is represented by a color gradient from light pink to red, where red probes have higher scores and are preferred over light red probes. The position of probes along the transcript is represented together with sequence features that can guide probe selection. Different graphs represent: maximum sequence identity of BLAST matches to other transcripts (blue top), maximum sequence identity of BLAST matches to a database of repetitive sequences (red); predicted melting temperature in a window of 20 residues (green); number of ESTs that skip an exon but include the exons flanking it (blue bottom). The actual melting temperature of probes is independently calculated by Primer3, while the purpose of the green graph is to give an indication on whether certain regions of the transcript have a melting temperature that is too low or too high to be amenable for probe design. Blue and red graphs represent sequence features that are not considered in the scoring of Primer3 probes. Secondary RNA structure is not considered for probe design and it is always recommended to simultaneously use multiple probe pairs that target different regions of a transcript to increase the sensitivity of the assay and to minimize experimental artifacts due to transcript accessibility. The software does not explicitly deal with alternative splicing. If one is interested in targeting a specific splice variant the best approach is to use the sequence of the isoform as input to the program. The aim of displaying exons that are skipped in publicly available EST data is to give the user the possibility to primarily target constitutive exons. Repetitive sequences should always be avoided during probe design since probe pairs can otherwise lead to unspecific signals.

Supplementary Figure 3 Specificity control experiments for PLAYR.

Detection of the ACTB transcript in Jurkat cells. No signal is detected when PLAYR is performed in absence of probes (NO PROBES), in absence of insert (NO INSERT), in absence of backbone (NO BACKBONE), in absence of ligase (NO LIGATION), in absence of detection oligo (NO DETECTION OLIGO), in presence of probes directed against the anti-sense ACTB transcript (SENSE PROBES), in presence of probes with the same half of the insert-complementary sequence (ORIENTATION CONTROL), or in presence of non-cognate probe pairs targeting different transcripts (ACTB and GAPDH, GENE-SPECIFICITY CONTROL). Signals were detected by flow cytometry. 4 probe pairs were used per gene.

Supplementary Figure 4 Detection of specific transcripts in single cells by flow cytometry using multiple probe pairs.

a) Detection of CD10 and CD3E by PLAYR in three replicates. Jurkat and NALM-6 cells were incubated with the indicated number of probe pairs and analyzed by flow cytometry. The expression of CD3E and CD10 is specific to Jurkat and NALM-6 cells respectively. b) The intensity of PLAYR signals depends on the distance between PLAYR probe binding sites on a target transcript. Multiple adjacent probe pairs spanning a transcript were designed and tested in all possible pairwise combinations. The x-axis represents the distance between each pair of probes, and the y-axis represents the ratio between the signal obtained with a given combination and the signal obtained with the corresponding adjacent cognate probe (i.e., the one that was originally designed to be used in the pair).

Source data

Supplementary Figure 5 Protocol optimization for RNA detection with simultaneous antibody staining.

Several surface antigens are disrupted by methanol permeabilization, which requires antibody staining to be performed before this step in the PLAYR protocol. In addition, we have found that crosslinking with the BS3 crosslinker is necessary to preserve antibody staining during the PLAYR protocol, as determined in multiple experiments with different antibodies (data not shown). However, RNA is progressively degraded during the incubations that are necessary to perform the steps mentioned above. This can be avoided by permeabilization of cells in the presence of RNAse inhibitors that inhibit endogenous and exogenous RNAses. Shown are signal intensities for the ACTB transcript in monocytes as measured by PLAYR when antibody staining is performed in different buffers containing RNAsin Ribonuclease Inhibitor (RNAsin) only or in combination with ribonucleoside vanadyl complex (RVC) and polyvinylsulfonic acid (PVS). BD Perm/Wash buffer is a commercially available saponin-based buffer and was replaced with PBS + 2% saponin that performed comparably. Signals obtained when cells are permeabilized directly without antibody staining are shown as a control. We decided to omit RVC in this step from the final protocol because the reagent interferes with the staining of some antibodies. Experiments were performed on a fluorescence flow cytometer with healthy PBMCs and monocytes were gated based on Forward and Side Scatter.

Supplementary Figure 6 Reproducibility of PLAYR measurements across technical replicates.

The plot displays the quantification of 22 different transcripts including negative controls across 16 technical replicates in mouse embryonic stem cells as measured by mass cytometry. Points represent mean values of replicates and bars indicate the full range of values for each transcript.

Source data

Supplementary Figure 7 Fluorescence flow cytometry gating strategy for human PBMCs.

Gating strategy used for human PBMC (see Fig. 4C and 4E of the main text).

Supplementary information

Supplementary Text and Figures

Supplementary Figures 1–7 (PDF 917 kb)

Supplementary Table 1

PLAYR probes and backbone-insert systems (XLSX 90 kb)

Supplementary Software

PLAYR Design software for probe design (ZIP 72 kb)

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Frei, A., Bava, FA., Zunder, E. et al. Highly multiplexed simultaneous detection of RNAs and proteins in single cells. Nat Methods 13, 269–275 (2016). https://doi.org/10.1038/nmeth.3742

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