MiRNA detection at single-cell resolution using microfluidic LNA flow-FISH

Methods Mol Biol. 2014;1211:245-60. doi: 10.1007/978-1-4939-1459-3_20.

Abstract

Flow cytometry in combination with fluorescent in situ hybridization (flow-FISH) is a powerful technique that can be utilized to rapidly detect nucleic acids at single-cell resolution without the need for homogenization or nucleic acid extraction. Here, we describe a microfluidic-based method which enables the detection of microRNAs or miRNAs in single intact cells by flow-FISH using locked nucleic acid (LNA)-containing probes. Our method can be applied to all RNA species including mRNA and small noncoding RNA and is suitable for multiplexing with protein immunostaining in the same cell. For demonstration of our method, this chapter details the detection of miR155 and CD69 protein in PMA and ionomycin-stimulated Jurkat cells. We also include instructions on how to set up a microfluidic chip sample preparation station to prepare cells for imaging and analysis on a commercial flow cytometer or a custom-built micro-flow cytometer.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, U.S. Gov't, Non-P.H.S.

MeSH terms

  • Antigens, CD / analysis
  • Antigens, Differentiation, T-Lymphocyte / analysis
  • Equipment Design
  • Flow Cytometry / instrumentation
  • Flow Cytometry / methods
  • Humans
  • Immunohistochemistry / methods
  • In Situ Hybridization, Fluorescence / instrumentation*
  • In Situ Hybridization, Fluorescence / methods
  • Jurkat Cells
  • Lectins, C-Type / analysis
  • MicroRNAs / analysis*
  • Microfluidic Analytical Techniques / instrumentation*
  • Microfluidic Analytical Techniques / methods
  • Nucleic Acid Amplification Techniques / instrumentation
  • Nucleic Acid Amplification Techniques / methods
  • Oligonucleotides / analysis*

Substances

  • Antigens, CD
  • Antigens, Differentiation, T-Lymphocyte
  • CD69 antigen
  • Lectins, C-Type
  • MIRN155 microRNA, human
  • MicroRNAs
  • Oligonucleotides
  • locked nucleic acid