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Analyze mRNA expression levels when antibody is limited

Flow cytometry with its ability to look at millions of cells, target multiplexing capabilities and straightforward workflow to detect both cell surface and intracellular proteins with single cell resolution has been the gold standard for the study of heterogeneous cell populations. However flow cytometry has historically been constrained by the availability and adequacy of antibodies to measure analytes. Non-coding RNA, viral transcripts, unique model organisms and targets or targets for which antibody development is troublesome have not been able to utilize the power of flow cytometry and have required numerous disconnected experiments to analyze their impact on cell subsets. PrimeFlow® RNA can be applied to detect target specific RNA for which available flow cytometry antibodies are nonexistent.

IL-23R Expression in Th17 Subset

Aims
Th17 cells are a subset of activated CD4+ T cells that are responsive to IL-1R1 and IL-23R signaling. Functionally, Th17 cells play a key role in host defense against extracellular microbes such as bacteria and fungi and play a significant role in autoimmune disease and its inflammatory response. The study of IL-23R has been hampered by the lack of antibodies with appropriate sensitivity; thereby most of our knowledge of IL-23R expression in Th17 cells has looked at gene expression at the population level with thousands or millions of cells analyzed in bulk. The data obtain has been unquestionably informative; however masks the actual heterogeneity. Here PrimeFlow® RNA is applied in combination with antibody staining to identify the heterogeneity of IL-23R in polarized Th17 cell subsets.

Results
Normal human peripheral blood cells that were cultured under Th17 polarizing conditions for 3 days and then restimulated showed a mix of Th17 cells that upregulated IL-23R mRNA and that did not upregulated IL-23R mRNA at that timepoint.

Conclusions
On strength of the PrimeFlow® RNA assay is the ability to detect most mRNAs in individual cells without being limited by antibody availability. This scan be applied to targets for which antibody development is troublesome (e.g. IL-23R, GPCRs, etc.), for unique model organisms (e.g. canine, fish, etc.), or for esoteric markers which no commercial antibodies are developed. This also presents the opportunity to detect non protein-coding RNA targets (lnc-RNA, viral transcripts, etc.) where antibodies cannot be created.

Figure 1: Detection of IL-23R in human Th17 polarized and expanded cells
Detection of IL-23R in human Th17 polarized and expanded cells

Legend: Normal human peripheral blood cells were cultured under Th17 polarizing conditions for 3 days, then restimulated with Cell Stimulation Cocktail (plus protein transport inhibitors) (cat. no. 00-4975) for 5 hours. Cells were labeled with Fixable Viablity Dye eFluor® 506 (cat. no. 65-0866) fixed and permeabilized using the PrimeFlow® RNA assay buffers, then intracellularly stained with anti-CD4 PE-eFluor® 610, anti-IL-17A PE-Cyanine7, and anti-IL-17AF eFluor® 450.