miRNA Detection at Single-Cell Resolution Using Microfluidic LNA Flow-FISH

  • Meiye Wu
  • Matthew E. Piccini
  • Anup K. SinghEmail author
Part of the Methods in Molecular Biology book series (MIMB, volume 1211)


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.

Key words

microRNA Locked nucleic acid Fluorescence in situ hybridization FISH Flow cytometry Multiplexing Single-cell resolution Microfluidics Rolling circle amplification 



The authors would like to thank Dr. Kit S. Lam of UC Davis for insightful discussions, Dr. Aarthi Chandrasakaran for critical reading of the manuscript, and Dr. Chung-yan Koh for providing Qdot reagents. The authors also thank Ron Renzi, JimVan De Vreugde, and Jim He for integration and automation of microfluidic platform. This work was supported by the following grants: R01 DE020891, funded by the NIDCR; the MISL Grand Challenge Laboratory Directed Research and Development program at Sandia National Laboratories; and P50GM085273 (the New Mexico Spatiotemporal Modeling Center) funded by the NIGMS.


  1. 1.
    Rufer N et al (1998) Telomere length dynamics in human lymphocyte subpopulations measured by flow cytometry. Nat Biotechnol 16(8): 743–747PubMedCrossRefGoogle Scholar
  2. 2.
    Silahtaroglu AN, Tommerup N, Vissing H (2003) FISHing with locked nucleic acids (LNA): evaluation of different LNA/DNA mixmers. Mol Cell Probes 17(4):165–169PubMedCrossRefGoogle Scholar
  3. 3.
    Kumar R et al (1998) The first analogues of LNA (locked nucleic acids): phosphorothioate-LNA and 2′-thio-LNA. Bioorg Med Chem Lett 8(16):2219–2222PubMedCrossRefGoogle Scholar
  4. 4.
    Robertson KL, Thach DC (2009) LNA flow-FISH: a flow cytometry-fluorescence in situ hybridization method to detect messenger RNA using locked nucleic acid probes. Anal Biochem 390(2):109–114PubMedCrossRefGoogle Scholar
  5. 5.
    Robertson KL et al (2010) Monitoring viral RNA in infected cells with LNA flow-FISH. RNA 16(8):1679–1685PubMedCrossRefPubMedCentralGoogle Scholar
  6. 6.
    Robertson KL, Vora GJ (2012) Locked nucleic acid and flow cytometry-fluorescence in situ hybridization for the detection of bacterial small noncoding RNAs. Appl Environ Microbiol 78(1):14–20PubMedCrossRefPubMedCentralGoogle Scholar
  7. 7.
    de Planell-Saguer M, Rodicio MC, Mourelatos Z (2010) Rapid in situ codetection of noncoding RNAs and proteins in cells and formalin-fixed paraffin-embedded tissue sections without protease treatment. Nat Protoc 5(6):1061–1073PubMedCrossRefGoogle Scholar
  8. 8.
    Wu M et al (2013) Single cell microRNA analysis using microfluidic flow cytometry. PLoS One 8(1):e55044PubMedCrossRefPubMedCentralGoogle Scholar
  9. 9.
    Wu M, Singh AK (2012) Single-cell protein analysis. Curr Opin Biotechnol 23(1):83–88PubMedCrossRefPubMedCentralGoogle Scholar
  10. 10.
    Wu M et al (2012) Microfluidically-unified cell culture, sample preparation, imaging and flow cytometry for measurement of cell signaling pathways with single cell resolution. Lab Chip 12(16):2823–2831PubMedCrossRefGoogle Scholar
  11. 11.
    Liu Y et al (2013) Single-cell measurements of IgE-mediated FcepsilonRI signaling using an integrated microfluidic platform. PLoS One 8(3):e60159PubMedCrossRefPubMedCentralGoogle Scholar
  12. 12.
    Yilmaz S, Singh AK (2012) Single cell genome sequencing. Curr Opin Biotechnol 23(3): 437–443PubMedCrossRefPubMedCentralGoogle Scholar
  13. 13.
    Liu P et al (2011) Microfluidic fluorescence in situ hybridization and flow cytometry (muFlowFISH). Lab Chip 11(16):2673–2679PubMedCrossRefPubMedCentralGoogle Scholar
  14. 14.
    Powell AA et al (2012) Single cell profiling of circulating tumor cells: transcriptional heterogeneity and diversity from breast cancer cell lines. PLoS One 7(5):e33788PubMedCrossRefPubMedCentralGoogle Scholar

Copyright information

© Springer Science+Business Media New York 2014

Authors and Affiliations

  1. 1.Biological Science and TechnologySandia National LaboratoriesLivermoreUSA

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