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

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 Reynolds, Brett Norris, Scott K. Mordecai, Quan Nguyen, Chunfai Lai and Daniel E. Kaufmann ()
Additional contact information
Filippos Porichis: The Ragon Institute of MGH, MIT and Harvard
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
Sara M. Miller: The Ragon Institute of MGH, MIT and Harvard
Damien Z. Soghoian: The Ragon Institute of MGH, MIT and Harvard
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

Nature Communications, 2014, vol. 5, issue 1, 1-12

Abstract: 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.

Date: 2014
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DOI: 10.1038/ncomms6641

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