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Analytical and Bioanalytical Chemistry

, Volume 409, Issue 14, pp 3497–3505 | Cite as

Amplification-free detection of microRNAs via a rapid microarray-based sandwich assay

  • Eoin Clancy
  • Martina Burke
  • Vahid Arabkari
  • Thomas Barry
  • Helena Kelly
  • Róisín M. Dwyer
  • Michael J. Kerin
  • Terry J. Smith
Paper in Forefront

Abstract

The detection and profiling of microRNAs are of great interest in disease diagnosis and prognosis. In this paper, we present a method for the rapid amplification-free detection of microRNAs from total RNA samples. In a two-step sandwich assay approach, fluorescently labeled reporter probes were first hybridized with their corresponding target microRNAs. The reaction mix was then added to a microarray to enable their specific capture and detection. Reporter probes were Tm equalized, enabling specificity by adjusting the length of the capture probe while maintaining the stabilizing effect brought about by coaxial base stacking. The optimized assay can specifically detect microRNAs in spiked samples at concentrations as low as 1 pM and from as little as 100 ng of total RNA in 2 h. The detection signal was linear between 1 and 100 pM (R2 = 0.99). Our assay data correlated well with results generated by qPCR when we profiled a select number of breast cancer related microRNAs in a total RNA sample.

Keywords

MicroRNA Microarray Sandwich Assay Amplification-free detection 

Notes

Acknowledgements

This work was supported by Science Foundation Ireland as part of the Biomedical Diagnostics Institute Centre for Science Excellence and Technology (10/CE/B1821).

Compliance with ethical standards

Conflict of interest

The authors declare that they have no conflict of interest.

Supplementary material

216_2017_298_MOESM1_ESM.pdf (129 kb)
ESM 1 (PDF 128 kb)

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Copyright information

© Springer-Verlag Berlin Heidelberg 2017

Authors and Affiliations

  • Eoin Clancy
    • 1
    • 2
  • Martina Burke
    • 1
    • 2
  • Vahid Arabkari
    • 1
    • 3
  • Thomas Barry
    • 4
  • Helena Kelly
    • 4
  • Róisín M. Dwyer
    • 5
  • Michael J. Kerin
    • 5
  • Terry J. Smith
    • 1
    • 2
  1. 1.Molecular Diagnostics Research Group, School of Natural Sciences and National Centre for Biomedical Engineering ScienceNUI GalwayGalwayIreland
  2. 2.Biomedical Diagnostics Institute Programme, National Centre for Biomedical Engineering ScienceNUI GalwayGalwayIreland
  3. 3.Discipline of Pathology, School of Medicine, Lambe Institute for Translational ResearchNUI GalwayGalwayIreland
  4. 4.Nucleic Acid Diagnostics Research Laboratory, Microbiology, School of Natural SciencesNUI GalwayGalwayIreland
  5. 5.Discipline of Surgery, School of Medicine, Lambe Institute for Translational ResearchNUI GalwayGalwayIreland

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