title: Simultaneous, Single-Cell Measurement of Messenger RNA, Cell Surface Proteins, and Intracellular Proteins.
abstract: Nucleic acid content can be quantified by flow cytometry through the use of intercalating compounds; however, measuring the presence of specific sequences has hitherto been difficult to achieve by this methodology. The primary obstacle to detecting discrete nucleic acid sequences by flow cytometry is their low quantity and the presence of high background signals, rendering the detection of hybridized fluorescent probes challenging. Amplification of nucleic acid sequences by molecular techniques such as in situ PCR have been applied to single-cell suspensions, but these approaches have not been easily adapted to conventional flow cytometry. An alternative strategy implements a Branched DNA technique, comprising target-specific probes and sequentially hybridized amplification reagents, resulting in a theoretical 8,000- to 16,000-fold increase in fluorescence signal amplification. The Branched DNA technique allows for the quantification of native and unmanipulated mRNA content with increased signal detection and reduced background. This procedure utilizes gentle fixation steps with low hybridization temperatures, leaving the assayed cells intact to permit their concomitant immunophenotyping. This technology has the potential to advance scientific discovery by correlating potentially small quantities of mRNA with many biological measurements at the single-cell level.
authors.affiliation: Department of Flow and Image Cytometry, Roswell Park Cancer Institute, Buffalo, New York.
authors.forename: Kah Teong
authors.initials: KT
authors.lastname: Soh
authors.affiliation: Department of Flow and Image Cytometry, Roswell Park Cancer Institute, Buffalo, New York.
authors.forename: Joseph D
authors.initials: JD
authors.lastname: Tario
authors.affiliation: Department of Flow and Image Cytometry, Roswell Park Cancer Institute, Buffalo, New York.
authors.forename: Sean
authors.initials: S
authors.lastname: Colligan
authors.affiliation: Department of Flow and Image Cytometry, Roswell Park Cancer Institute, Buffalo, New York.
authors.forename: Orla
authors.initials: O
authors.lastname: Maguire
authors.affiliation: Department of Flow and Image Cytometry, Roswell Park Cancer Institute, Buffalo, New York.
authors.forename: Dalin
authors.initials: D
authors.lastname: Pan
authors.affiliation: Department of Flow and Image Cytometry, Roswell Park Cancer Institute, Buffalo, New York.
authors.forename: Hans
authors.initials: H
authors.lastname: Minderman
authors.affiliation: Department of Flow and Image Cytometry, Roswell Park Cancer Institute, Buffalo, New York.
authors.forename: Paul K
authors.initials: PK
authors.lastname: Wallace
mesh_terms: D015703:Antigens, CD / Q000032:analysis*; D017209:Apoptosis; D018414:CD8-Positive T-Lymphocytes / Q000166:cytology; D002453:Cell Cycle; D002462:Cell Membrane / Q000378:metabolism*; D019295:Computational Biology / Q000379:methods; D004247:DNA / Q000032:analysis; D005434:Flow Cytometry / Q000379:methods*; D006801:Humans; D016130:Immunophenotyping / Q000379:methods; D017403:In Situ Hybridization; D007963:Leukocytes, Mononuclear / Q000166:cytology; D008565:Membrane Proteins / Q000032:analysis*; D009693:Nucleic Acid Hybridization; D010766:Phosphorylation; D016133:Polymerase Chain Reaction; D012333:RNA, Messenger / Q000032:analysis* / Q000378:metabolism; D013696:Temperature
chemical_list: D015703:Antigens, CD; D008565:Membrane Proteins; D012333:RNA, Messenger; D004247:DNA
keywords: Branched DNA; flow cytometry; in situ hybridization; leukocytes; mRNA sequence; target probe; transcription factors