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


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.

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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.

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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.

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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.

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Correspondence to Sanjay Tyagi.

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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.

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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).

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