title: Single cell microRNA analysis using microfluidic flow cytometry.
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.
authors: Department of Biotechnology and Bioengineering, Sandia National Laboratory, Livermore, CA, USA.
authors: Meiye
authors: M
authors: Wu
authors: Matthew
authors: M
authors: Piccini
authors: Chung-Yan
authors: CY
authors: Koh
authors: Kit S
authors: KS
authors: Lam
authors: Anup K
authors: AK
authors: Singh
mesh_terms: D015703:Antigens, CD / Q000378:metabolism; D000945:Antigens, Differentiation, T-Lymphocyte / Q000378:metabolism; D002454:Cell Differentiation / Q000187:drug effects; D049109:Cell Proliferation / Q000187:drug effects; D004867:Equipment Design; D005434:Flow Cytometry / Q000295:instrumentation*; D006801:Humans; D015759:Ionomycin / Q000494:pharmacology; D019169:Jurkat Cells; D037181:Lectins, C-Type / Q000378:metabolism; D008213:Lymphocyte Activation / Q000187:drug effects; D035683:MicroRNAs / Q000235:genetics*; D046210:Microfluidic Analytical Techniques / Q000295:instrumentation / Q000379:methods*; D059010:Single-Cell Analysis / Q000295:instrumentation*; D013601:T-Lymphocytes / Q000166:cytology / Q000187:drug effects / Q000276:immunology / Q000378:metabolism; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology; D015854:Up-Regulation / Q000187:drug effects; D000099192:CD69 Antigens
chemical_list: D015703:Antigens, CD; D000945:Antigens, Differentiation, T-Lymphocyte; D015759:Ionomycin; D037181:Lectins, C-Type; D035683:MicroRNAs; D013755:Tetradecanoylphorbol Acetate; D000099192:CD69 Antigens; C524942:MIRN155 microRNA, human
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