Abstract
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.
Publication types
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Research Support, N.I.H., Extramural
MeSH terms
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Antigens, CD / metabolism
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Antigens, Differentiation, T-Lymphocyte / metabolism
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Cell Differentiation / drug effects
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Cell Proliferation / drug effects
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Equipment Design
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Flow Cytometry / instrumentation*
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Humans
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Ionomycin / pharmacology
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Jurkat Cells
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Lectins, C-Type / metabolism
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Lymphocyte Activation / drug effects
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MicroRNAs / genetics*
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Microfluidic Analytical Techniques / instrumentation
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Microfluidic Analytical Techniques / methods*
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Single-Cell Analysis / instrumentation*
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T-Lymphocytes / cytology
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T-Lymphocytes / drug effects
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T-Lymphocytes / immunology
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T-Lymphocytes / metabolism
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Tetradecanoylphorbol Acetate / pharmacology
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Up-Regulation / drug effects
Substances
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Antigens, CD
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Antigens, Differentiation, T-Lymphocyte
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CD69 antigen
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Lectins, C-Type
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MIRN155 microRNA, human
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MicroRNAs
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Ionomycin
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Tetradecanoylphorbol Acetate