title: High-throughput detection of miRNAs and gene-specific mRNA at the single-cell level by flow cytometry.
abstract: Fluorescent in situ hybridization (FISH) is a method that uses fluorescent probes to detect specific nucleic acid sequences at the single-cell level. Here we describe optimized protocols that exploit a highly sensitive FISH method based on branched DNA technology to detect mRNA and miRNA in human leukocytes. This technique can be multiplexed and combined with fluorescent antibody protein staining to address a variety of questions in heterogeneous cell populations. We demonstrate antigen-specific upregulation of IFNγ and IL-2 mRNAs in HIV- and CMV-specific T cells. We show simultaneous detection of cytokine mRNA and corresponding protein in single cells. We apply this method to detect mRNAs for which flow antibodies against the corresponding proteins are poor or are not available. We use this technique to show modulation of a microRNA critical for T-cell function, miR-155. We adapt this assay for simultaneous detection of mRNA and proteins by ImageStream technology.
authors.affiliation: 1] The Ragon Institute of MGH, MIT and Harvard, Cambridge, Massachusetts 02139, USA [2] Center for HIV/AIDS Vaccine Immunology and Immunogen Discovery.
authors.forename: Filippos
authors.initials: F
authors.lastname: Porichis
authors.affiliation: The Ragon Institute of MGH, MIT and Harvard, Cambridge, Massachusetts 02139, USA.
authors.forename: Meghan G
authors.initials: MG
authors.lastname: Hart
authors.affiliation: The Ragon Institute of MGH, MIT and Harvard, Cambridge, Massachusetts 02139, USA.
authors.forename: Morgane
authors.initials: M
authors.lastname: Griesbeck
authors.affiliation: The Ragon Institute of MGH, MIT and Harvard, Cambridge, Massachusetts 02139, USA.
authors.forename: Holly L
authors.initials: HL
authors.lastname: Everett
authors.affiliation: The Ragon Institute of MGH, MIT and Harvard, Cambridge, Massachusetts 02139, USA.
authors.forename: Muska
authors.initials: M
authors.lastname: Hassan
authors.affiliation: Centre de Recherche du Centre Hospitalier de l'Université de Montréal (CRCHUM) and University of Montreal, Montréal, Quebec, Canada H2X 0A9.
authors.forename: Amy E
authors.initials: AE
authors.lastname: Baxter
authors.affiliation: 1] The Ragon Institute of MGH, MIT and Harvard, Cambridge, Massachusetts 02139, USA [2] Center for HIV/AIDS Vaccine Immunology and Immunogen Discovery.
authors.forename: Madelene
authors.initials: M
authors.lastname: Lindqvist
authors.affiliation: The Ragon Institute of MGH, MIT and Harvard, Cambridge, Massachusetts 02139, USA.
authors.forename: Sara M
authors.initials: SM
authors.lastname: Miller
authors.affiliation: 1] The Ragon Institute of MGH, MIT and Harvard, Cambridge, Massachusetts 02139, USA [2] Center for HIV/AIDS Vaccine Immunology and Immunogen Discovery.
authors.forename: Damien Z
authors.initials: DZ
authors.lastname: Soghoian
authors.affiliation: The Ragon Institute of MGH, MIT and Harvard, Cambridge, Massachusetts 02139, USA.
authors.forename: Daniel G
authors.initials: DG
authors.lastname: Kavanagh
authors.affiliation: Affymetrix, Inc., 3380 Central Expressway, Santa Clara, California 95051, USA.
authors.forename: Susan
authors.initials: S
authors.lastname: Reynolds
authors.affiliation: Affymetrix, Inc., 3380 Central Expressway, Santa Clara, California 95051, USA.
authors.forename: Brett
authors.initials: B
authors.lastname: Norris
authors.affiliation: Department of Pathology, Massachusetts General Hospital, Boston, Massachusetts 02114, USA.
authors.forename: Scott K
authors.initials: SK
authors.lastname: Mordecai
authors.affiliation: Affymetrix, Inc., 3380 Central Expressway, Santa Clara, California 95051, USA.
authors.forename: Quan
authors.initials: Q
authors.lastname: Nguyen
authors.affiliation: Affymetrix, Inc., 3380 Central Expressway, Santa Clara, California 95051, USA.
authors.forename: Chunfai
authors.initials: C
authors.lastname: Lai
authors.affiliation: 1] The Ragon Institute of MGH, MIT and Harvard, Cambridge, Massachusetts 02139, USA [2] Center for HIV/AIDS Vaccine Immunology and Immunogen Discovery [3] Centre de Recherche du Centre Hospitalier de l'Université de Montréal (CRCHUM) and University of Montreal, Montréal, Quebec, Canada H2X 0A9.
authors.forename: Daniel E
authors.initials: DE
authors.lastname: Kaufmann
mesh_terms: D003587:Cytomegalovirus / Q000276:immunology; D005434:Flow Cytometry / Q000379:methods*; D006678:HIV / Q000276:immunology; D015658:HIV Infections / Q000276:immunology; D006801:Humans; D017404:In Situ Hybridization, Fluorescence / Q000379:methods*; D007371:Interferon-gamma / Q000235:genetics; D007376:Interleukin-2 / Q000235:genetics; D035683:MicroRNAs / Q000032:analysis* / Q000235:genetics; D012333:RNA, Messenger / Q000032:analysis*; D013601:T-Lymphocytes / Q000166:cytology / Q000276:immunology / Q000378:metabolism*; D015854:Up-Regulation
chemical_list: C508594:IL2 protein, human; D007376:Interleukin-2; C524942:MIRN155 microRNA, human; D035683:MicroRNAs; D012333:RNA, Messenger; D007371:Interferon-gamma