title: Single-cell Gene Expression Profiling Using FACS and qPCR with Internal Standards.
abstract: Gene expression measurements from bulk populations of cells can obscure the considerable transcriptomic variation of individual cells within those populations. Single-cell gene expression measurements can help assess the role of noise in gene expression, identify correlations in the expression of pairs of genes, and reveal subpopulations of cells that respond differently to a stimulus. Here, we describe a procedure to measure the expression of up to 96 genes in single mammalian cells isolated from a population growing in tissue culture. Cells are sorted into lysis buffer by fluorescence-activated cell sorting (FACS), and the mRNA species of interest are reverse-transcribed and amplified. Gene expression is then measured using a microfluidic real-time PCR machine, which performs up to 96 qPCR assays on up to 96 samples at a time. We also describe the generation and use of PCR amplicon standards to enable the estimation of the absolute number of each transcript. Compared with other methods of measuring gene expression in single cells, this approach allows for the quantification of more distinct transcripts than RNA FISH at a lower cost than RNA-Seq.
authors.affiliation: Laboratory of Pathology, Center for Cancer Research, National Cancer Institute.
authors.forename: Joshua R
authors.initials: JR
authors.lastname: Porter
authors.affiliation: Experimental Transplantation and Immunology Branch, Center for Cancer Research, National Cancer Institute.
authors.forename: William G
authors.initials: WG
authors.lastname: Telford
authors.affiliation: Laboratory of Pathology, Center for Cancer Research, National Cancer Institute; eric.batchelor@nih.gov.
authors.forename: Eric
authors.initials: E
authors.lastname: Batchelor
mesh_terms: D000818:Animals; D005434:Flow Cytometry / Q000379:methods*; D020869:Gene Expression Profiling / Q000379:methods*; D012333:RNA, Messenger / Q000235:genetics*; D060888:Real-Time Polymerase Chain Reaction; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods*; D059467:Transcriptome
chemical_list: D012333:RNA, Messenger