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

Abstract

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, Reprints and permissions information is available at www.nature.com/reprints.

Multiplexed detection and isolation of viable low-frequency cytokine-secreting human B cells using cytokine secretion assay and flow cytometry (CSA-Flow)

“…The assay relies on sequential antibody-based cell surface labelling, thereby preserving cell integrity and minimizing cell loss 34,35 . These features, along with the ability to isolate viable and functional low frequency cytokine-defined cells, encapsulate the main competitive advantage of this assay over traditional intracellular cytokine-staining or flow fluorescent in situ hybridization [19][20][21][22][23][24][25] . Assay multiplexing is attractive for maximizing the output of potentially limited clinical sample volumes, and has been previously used with CSA to both isolate and distinguish distinct cytokine-secreting immune cells 42,43 .…”

“…impact of such cells on responses of other cells in vitro, or when introduced in vivo (potentially in the form of adoptive cell transfer 18 ). However, the isolation of viable and purified cytokine-expressing immune cells has presented a technical challenge, as commonly used intracellular cytokine-staining (ICS) or flow-fluorescent in-situ hybridization approaches involve cell fixation and permeabilization [19][20][21][22][23][24][25] . The resultant loss of cell viability and integrity seriously limits the use of cells for comprehensive functional profiling and downstream applications [19][20][21][22][23][24][25] .…”

“…However, the isolation of viable and purified cytokine-expressing immune cells has presented a technical challenge, as commonly used intracellular cytokine-staining (ICS) or flow-fluorescent in-situ hybridization approaches involve cell fixation and permeabilization [19][20][21][22][23][24][25] . The resultant loss of cell viability and integrity seriously limits the use of cells for comprehensive functional profiling and downstream applications [19][20][21][22][23][24][25] . While viable cells can be sorted based on certain cell surface-markers that have been reported to enrich for particular cytokine-expressing immune cells, such markers are inevitably neither fully sensitive nor specific [26][27][28][29][30][31][32][33] .…”

Multidimensional analysis of Gammaherpesvirus RNA expression reveals unexpected heterogeneity of gene expression

“…FMO) control to accurately define background and signal to noise ratio for targets of interest. We consider multiparameter flow cytometry, as well as mass cytometry [32, 33], two increasingly useful technologies to afford new insight into heterogeneity of virus infection and gene expression that provide a complementary approach to other single cell technologies.…”

Single-Cell Technologies Applied to HIV-1 Research: Reaching Maturity

“…Identification of HIV-1 + cells is typically achieved by the detection of viral RNA (vRNA), viral DNA (vDNA) or expression of the structural protein p24. Several direct single-cell virus detection imaging methods with signal amplification were developed in the past years, including in situ PCR (Bagasra et al, 1993), tyramide amplification (Soontornniyomkij et al, 1999), and the tunable rolling circle amplification (Frei et al, 2016;Duckworth et al, 2019). All these methods relied on sensitive RNA or DNA fluorescence detection through signal amplification, but at the cost of low reproducibility and high false detection rate due to high background.…”