biostudies-literatureDominguez MHIntramural NIH HHSBill & Melinda Gates FoundationIntramural Research Program of the NIAID, NIH133-45https://www.ebi.ac.uk/biostudies/studies/S-EPMC3814038biostudies-literatureUnknown391(1-2)Highly multiplexed, single-cell technologies reveal important heterogeneity within cell populations. Recently, technologies to simultaneously measure expression of 96 (or more) genes from a single cell have been developed for immunologic monitoring. Here, we report a rigorous, optimized, quantitative methodology for using this technology. Specifically: we describe a unique primer/probe qualification method necessary for quantitative results; we show that primers do not compete in highly multiplexed amplifications; we define the limit of detection for this assay as a single mRNA transcript; and, we show that the technical reproducibility of the system is very high. We illustrate two disparate applications of the platform: a "bulk" approach that measures expression patterns from 100 cells atJournal of immunological methodsHighly multiplexed quantitation of gene expression on single cells.PMC3814038Z99 AI999999OPP1032325Dominguez MHFinak GKoup RAMa SChattopadhyay PKRoederer MGottardo RMcDavid ALamoreaux LfalseHighly multiplexed quantitation of gene expression on single cells.Highly multiplexed, single-cell technologies reveal important heterogeneity within cell populations. Recently, technologies to simultaneously measure expression of 96 (or more) genes from a single cell have been developed for immunologic monitoring. Here, we report a rigorous, optimized, quantitative methodology for using this technology. Specifically: we describe a unique primer/probe qualification method necessary for quantitative results; we show that primers do not compete in highly multiplexed amplifications; we define the limit of detection for this assay as a single mRNA transcript; and, we show that the technical reproducibility of the system is very high. We illustrate two disparate applications of the platform: a "bulk" approach that measures expression patterns from 100 cells at2013-01-01T00:00:00Z2013 May2025-04-29T11:00:51.376Z2019-03-27T01:17:53ZS-EPMC38140382350078110.1016/j.jim.2013.03.002