Single Cell MicroRNA Analysis Using Microfluidic Flow Cytometry

MicroRNAs (miRNAs) are non-coding small RNAs that have cell type and cell context-dependent expression and function. To study miRNAs at single-cell resolution, we have developed a novel microfluidic approach, where flow fluorescent in situ hybridization (flow-FISH) using locked-nucleic acid probes is combined with rolling circle amplification to detect the presence and localization of miRNA. Furthermore, our flow cytometry approach allows analysis of gene-products potentially targeted by miRNA together with the miRNA in the same cells. We demonstrate simultaneous measurement of miR155 and CD69 in 12-O-tetradecanoylphorbol 13-acetate (PMA) and Ionomycin stimulated Jurkat cells. The flow-FISH method can be completed in ∼10 h, utilizes only 170 nL of reagent per experimental condition, and is the first to directly detect miRNA in single cells using flow cytometry.

[1]  C. Perou,et al.  A custom microarray platform for analysis of microRNA gene expression , 2004, Nature Methods.

[2]  O. Söderberg,et al.  In Situ Proximity Ligation Assay for Microscopy and Flow Cytometry , 2011, Current protocols in cytometry.

[3]  Z. Mourelatos,et al.  Rapid in situ codetection of noncoding RNAs and proteins in cells and formalin-fixed paraffin-embedded tissue sections without protease treatment , 2010, Nature Protocols.

[4]  Yooli K. Light,et al.  Microfluidic fluorescence in situ hybridization and flow cytometry (μFlowFISH). , 2011, Lab on a chip.

[5]  N. Rajewsky,et al.  Regulation of the Germinal Center Response by MicroRNA-155 , 2007, Science.

[6]  Pilar Martín,et al.  CD69 Association with Jak3/Stat5 Proteins Regulates Th17 Cell Differentiation , 2010, Molecular and Cellular Biology.

[7]  M. Stoffel,et al.  MicroRNAs: a new class of regulatory genes affecting metabolism. , 2006, Cell metabolism.

[8]  Sam Griffiths-Jones,et al.  The microRNA Registry , 2004, Nucleic Acids Res..

[9]  B. Nielsen.  MicroRNA in situ hybridization. , 2012, Methods in molecular biology.

[10]  E. Miska,et al.  MicroRNA functions in animal development and human disease , 2005, Development.

[11]  F. Slack,et al.  MicroRNAs en route to the clinic: progress in validating and targeting microRNAs for cancer therapy , 2011, Nature Reviews Cancer.

[12]  Arthur Weiss,et al.  The role of T3 surface molecules in the activation of human T cells: a two-stimulus requirement for IL 2 production reflects events occurring at a pre-translational level. , 1984, Journal of immunology.

[13]  U. Landegren,et al.  Proximity ligation assays for sensitive and specific protein analyses. , 2005, Analytical biochemistry.

[14]  Nóra Varga,et al.  Sensitive and specific detection of microRNAs by northern blot analysis using LNA-modified oligonucleotide probes. , 2004, Nucleic acids research.

[15]  Caroline Lee,et al.  220-plex microRNA expression profile of a single cell , 2006, Nature Protocols.

[16]  A. Singh,et al.  Single Cell Protein Analysis , 2015, Methods in Molecular Biology.

[17]  A. Singh,et al.  Single cell genome sequencing. , 2012, Current opinion in biotechnology.

[18]  S. Freier,et al.  Absolute quantitation of microRNAs with a PCR-based assay. , 2006, Analytical biochemistry.

[19]  Thomas D. Perroud,et al.  Microfluidically-unified cell culture, sample preparation, imaging and flow cytometry for measurement of cell signaling pathways with single cell resolution. , 2012, Lab on a chip.

[20]  Ryan M. O’Connell,et al.  MicroRNAs: new regulators of immune cell development and function , 2008, Nature Immunology.

[21]  Vladimir Benes,et al.  A sensitive array for microRNA expression profiling (miChip) based on locked nucleic acids (LNA). , 2006, RNA.

[22]  G. Illei,et al.  MicroRNAs in Sjögren's syndrome as a prototypic autoimmune disease. , 2010, Autoimmunity reviews.

[23]  Hana Lee,et al.  Foxp3-dependent microRNA155 confers competitive fitness to regulatory T cells by targeting SOCS1 protein. , 2009, Immunity.

[24]  Gyan Bhanot,et al.  Single Cell Profiling of Circulating Tumor Cells: Transcriptional Heterogeneity and Diversity from Breast Cancer Cell Lines , 2012, PloS one.

[25]  J. Mauël,et al.  CD69 and regulation of the immune function. , 1999, Immunopharmacology and immunotoxicology.

[26]  Nan Deng,et al.  Transcriptome and targetome analysis in MIR155 expressing cells using RNA-seq. , 2010, RNA.

[27]  M. Zavolan,et al.  miRNA in situ hybridization in formaldehyde and EDC–fixed tissues , 2009, Nature Methods.

[28]  F. Tang,et al.  MicroRNA expression profiling of single whole embryonic stem cells , 2006, Nucleic acids research.

[29]  Jonas Jarvius,et al.  Cytokine detection by antibody-based proximity ligation. , 2004, Proceedings of the National Academy of Sciences of the United States of America.

[30]  Brian S. Roberts,et al.  Simple, quantitative primer-extension PCR assay for direct monitoring of microRNAs and short-interfering RNAs. , 2005, RNA.

[31]  H. Lodish,et al.  Micromanagement of the immune system by microRNAs , 2008, Nature Reviews Immunology.