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


Department of Molecular and Clinical Cancer Medicine, Institute of Translational Medicine, University of Liverpool, Liverpool, UK. [Email]


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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