title: Highly multiplexed quantitation of gene expression on single cells.
abstract: 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 at a time in high throughput to define gene signatures, and a single-cell approach to define the coordinate expression patterns of multiple genes and reveal unique subsets of cells.
authors.affiliation: ImmunoTechnology Section, Vaccine Research Center, NIAID, NIH, United States.
authors.forename: Maria H
authors.initials: MH
authors.lastname: Dominguez
authors.forename: Pratip K
authors.initials: PK
authors.lastname: Chattopadhyay
authors.forename: Steven
authors.initials: S
authors.lastname: Ma
authors.forename: Laurie
authors.initials: L
authors.lastname: Lamoreaux
authors.forename: Andrew
authors.initials: A
authors.lastname: McDavid
authors.forename: Greg
authors.initials: G
authors.lastname: Finak
authors.forename: Raphael
authors.initials: R
authors.lastname: Gottardo
authors.forename: Richard A
authors.initials: RA
authors.lastname: Koup
authors.forename: Mario
authors.initials: M
authors.lastname: Roederer
mesh_terms: D000818:Animals; D017931:DNA Primers; D020869:Gene Expression Profiling / Q000379:methods*; D005786:Gene Expression Regulation; D005819:Genetic Markers; D057166:High-Throughput Screening Assays / Q000379:methods*; D006801:Humans; D057230:Limit of Detection; D016014:Linear Models; D008213:Lymphocyte Activation / Q000235:genetics*; D016131:Lymphocyte Subsets / Q000276:immunology*; D008253:Macaca mulatta; D060885:Multiplex Polymerase Chain Reaction; D012333:RNA, Messenger / Q000378:metabolism*; D015203:Reproducibility of Results; D020133:Reverse Transcriptase Polymerase Chain Reaction; D013601:T-Lymphocytes / Q000276:immunology*
chemical_list: D017931:DNA Primers; D005819:Genetic Markers; D012333:RNA, Messenger