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

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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.

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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).

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Authors and Affiliations

  1. Molecular Diagnostics Research Group, School of Natural Sciences and National Centre for Biomedical Engineering Science, NUI Galway, Galway, H91 CF50, Ireland

    Eoin Clancy, Martina Burke, Vahid Arabkari & Terry J. Smith

  2. Biomedical Diagnostics Institute Programme, National Centre for Biomedical Engineering Science, NUI Galway, Galway, H91 CF50, Ireland

    Eoin Clancy, Martina Burke & Terry J. Smith

  3. Discipline of Pathology, School of Medicine, Lambe Institute for Translational Research, NUI Galway, Galway, H91 CF50, Ireland

    Vahid Arabkari

  4. Nucleic Acid Diagnostics Research Laboratory, Microbiology, School of Natural Sciences, NUI Galway, Galway, H91 CF50, Ireland

    Thomas Barry & Helena Kelly

  5. Discipline of Surgery, School of Medicine, Lambe Institute for Translational Research, NUI Galway, Galway, H91 CF50, Ireland

    Róisín M. Dwyer & Michael J. Kerin

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  1. Eoin Clancy
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  2. Martina Burke
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Correspondence to Eoin Clancy.

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Clancy, E., Burke, M., Arabkari, V. et al. Amplification-free detection of microRNAs via a rapid microarray-based sandwich assay. Anal Bioanal Chem 409, 3497–3505 (2017). https://doi.org/10.1007/s00216-017-0298-6

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  • DOI: https://doi.org/10.1007/s00216-017-0298-6

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