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 i...

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Bibliographic Details
Published in:PloS one 2013-01, Vol.8 (1), p.e55044
Main Authors: Wu, Meiye, Piccini, Matthew, Koh, Chung-Yan, Lam, Kit S, Singh, Anup K
Format: Article
Language:English
Subjects:
Acetic acid
Antigens, CD - metabolism
Antigens, Differentiation, T-Lymphocyte - metabolism
BASIC BIOLOGICAL SCIENCES
Biochemistry
Bioengineering
Biology
Biotechnology
CD69 antigen
Cell culture
Cell Differentiation - drug effects
Cell Proliferation - drug effects
Cytogenetics
Cytokines
Cytometry
Equipment Design
Fishes
Flow cytometry
Flow Cytometry - instrumentation
Fluorescence
Fluorescence in situ hybridization
Fluorescent indicators
Gene expression
Humans
Hybridization
Ionomycin
Ionomycin - pharmacology
Jurkat Cells
Laboratories
Lectins, C-Type - metabolism
Localization
Lymphocyte Activation - drug effects
Lymphocytes
Microfluidic Analytical Techniques - instrumentation
Microfluidic Analytical Techniques - methods
Microfluidics
MicroRNA
MicroRNAs
MicroRNAs - genetics
Microscopy
miRNA
Molecular biology
Proteins
Ribonucleic acid
RNA
Science & Technology - Other Topics
Single-Cell Analysis - instrumentation
Stem cells
T-Lymphocytes - cytology
T-Lymphocytes - drug effects
T-Lymphocytes - immunology
T-Lymphocytes - metabolism
Tetradecanoylphorbol Acetate - pharmacology
Up-Regulation - drug effects
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Description
Summary: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.
ISSN:1932-6203
1932-6203
DOI:10.1371/journal.pone.0055044