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High-throughput detection of miRNAs and gene-specific mRNA at the single-cell level by flow cytometry

Author

Listed:
  • Filippos Porichis

    (The Ragon Institute of MGH, MIT and Harvard
    Center for HIV/AIDS Vaccine Immunology and Immunogen Discovery)

  • Meghan G. Hart

    (The Ragon Institute of MGH, MIT and Harvard)

  • Morgane Griesbeck

    (The Ragon Institute of MGH, MIT and Harvard)

  • Holly L. Everett

    (The Ragon Institute of MGH, MIT and Harvard)

  • Muska Hassan

    (The Ragon Institute of MGH, MIT and Harvard)

  • Amy E. Baxter

    (Centre de Recherche du Centre Hospitalier de l'Université de Montréal (CRCHUM) and University of Montreal)

  • Madelene Lindqvist

    (The Ragon Institute of MGH, MIT and Harvard
    Center for HIV/AIDS Vaccine Immunology and Immunogen Discovery)

  • Sara M. Miller

    (The Ragon Institute of MGH, MIT and Harvard)

  • Damien Z. Soghoian

    (The Ragon Institute of MGH, MIT and Harvard
    Center for HIV/AIDS Vaccine Immunology and Immunogen Discovery)

  • Daniel G. Kavanagh

    (The Ragon Institute of MGH, MIT and Harvard)

  • Susan Reynolds

    (Affymetrix, Inc., 3380 Central Expressway)

  • Brett Norris

    (Affymetrix, Inc., 3380 Central Expressway)

  • Scott K. Mordecai

    (Massachusetts General Hospital)

  • Quan Nguyen

    (Affymetrix, Inc., 3380 Central Expressway)

  • Chunfai Lai

    (Affymetrix, Inc., 3380 Central Expressway)

  • Daniel E. Kaufmann

    (The Ragon Institute of MGH, MIT and Harvard
    Center for HIV/AIDS Vaccine Immunology and Immunogen Discovery
    Centre de Recherche du Centre Hospitalier de l'Université de Montréal (CRCHUM) and University of Montreal)

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.

Suggested Citation

  • Filippos Porichis & Meghan G. Hart & Morgane Griesbeck & Holly L. Everett & Muska Hassan & Amy E. Baxter & Madelene Lindqvist & Sara M. Miller & Damien Z. Soghoian & Daniel G. Kavanagh & Susan Reynold, 2014. "High-throughput detection of miRNAs and gene-specific mRNA at the single-cell level by flow cytometry," Nature Communications, Nature, vol. 5(1), pages 1-12, December.
    • Handle: RePEc:nat:natcom:v:5:y:2014:i:1:d:10.1038_ncomms6641
    DOI: 10.1038/ncomms6641
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    Cited by:

    1. Timon Wittenstein & Nava Leibovich & Andreas Hilfinger, 2022. "Quantifying biochemical reaction rates from static population variability within incompletely observed complex networks," PLOS Computational Biology, Public Library of Science, vol. 18(6), pages 1-21, June.

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