Single cell microRNA analysis using microfluidic flow cytometry

PLoS One. 2013;8(1):e55044. doi: 10.1371/journal.pone.0055044. Epub 2013 Jan 30.

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

  • Research Support, N.I.H., Extramural

MeSH terms

  • Antigens, CD / metabolism
  • Antigens, Differentiation, T-Lymphocyte / metabolism
  • Cell Differentiation / drug effects
  • Cell Proliferation / drug effects
  • Equipment Design
  • Flow Cytometry / instrumentation*
  • Humans
  • Ionomycin / pharmacology
  • Jurkat Cells
  • Lectins, C-Type / metabolism
  • Lymphocyte Activation / drug effects
  • MicroRNAs / genetics*
  • Microfluidic Analytical Techniques / instrumentation
  • Microfluidic Analytical Techniques / methods*
  • Single-Cell Analysis / instrumentation*
  • T-Lymphocytes / cytology
  • T-Lymphocytes / drug effects
  • T-Lymphocytes / immunology
  • T-Lymphocytes / metabolism
  • Tetradecanoylphorbol Acetate / pharmacology
  • Up-Regulation / drug effects

Substances

  • Antigens, CD
  • Antigens, Differentiation, T-Lymphocyte
  • CD69 antigen
  • Lectins, C-Type
  • MIRN155 microRNA, human
  • MicroRNAs
  • Ionomycin
  • Tetradecanoylphorbol Acetate