title: Amplification-free detection of microRNAs via a rapid microarray-based sandwich assay.
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 Tmequalized, 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.
authors.affiliation: Molecular Diagnostics Research Group, School of Natural Sciences and National Centre for Biomedical Engineering Science, NUI Galway, Galway, H91 CF50, Ireland. eoin.clancy@nuigalway.ie.|Biomedical Diagnostics Institute Programme, National Centre for Biomedical Engineering Science, NUI Galway, Galway, H91 CF50, Ireland. eoin.clancy@nuigalway.ie.
authors.forename: Eoin
authors.initials: E
authors.lastname: Clancy
authors.affiliation: Molecular Diagnostics Research Group, School of Natural Sciences and National Centre for Biomedical Engineering Science, NUI Galway, Galway, H91 CF50, Ireland.|Biomedical Diagnostics Institute Programme, National Centre for Biomedical Engineering Science, NUI Galway, Galway, H91 CF50, Ireland.
authors.forename: Martina
authors.initials: M
authors.lastname: Burke
authors.affiliation: Molecular Diagnostics Research Group, School of Natural Sciences and National Centre for Biomedical Engineering Science, NUI Galway, Galway, H91 CF50, Ireland.|Discipline of Pathology, School of Medicine, Lambe Institute for Translational Research, NUI Galway, Galway, H91 CF50, Ireland.
authors.forename: Vahid
authors.initials: V
authors.lastname: Arabkari
authors.affiliation: Nucleic Acid Diagnostics Research Laboratory, Microbiology, School of Natural Sciences, NUI Galway, Galway, H91 CF50, Ireland.
authors.forename: Thomas
authors.initials: T
authors.lastname: Barry
authors.affiliation: Nucleic Acid Diagnostics Research Laboratory, Microbiology, School of Natural Sciences, NUI Galway, Galway, H91 CF50, Ireland.
authors.forename: Helena
authors.initials: H
authors.lastname: Kelly
authors.affiliation: Discipline of Surgery, School of Medicine, Lambe Institute for Translational Research, NUI Galway, Galway, H91 CF50, Ireland.
authors.forename: Róisín M
authors.initials: RM
authors.lastname: Dwyer
authors.affiliation: Discipline of Surgery, School of Medicine, Lambe Institute for Translational Research, NUI Galway, Galway, H91 CF50, Ireland.
authors.forename: Michael J
authors.initials: MJ
authors.lastname: Kerin
authors.affiliation: Molecular Diagnostics Research Group, School of Natural Sciences and National Centre for Biomedical Engineering Science, NUI Galway, Galway, H91 CF50, Ireland.|Biomedical Diagnostics Institute Programme, National Centre for Biomedical Engineering Science, NUI Galway, Galway, H91 CF50, Ireland.
authors.forename: Terry J
authors.initials: TJ
authors.lastname: Smith
mesh_terms: D005456:Fluorescent Dyes / Q000737:chemistry; D006801:Humans; D057230:Limit of Detection; D035683:MicroRNAs / Q000032:analysis*; D009693:Nucleic Acid Hybridization / Q000379:methods*; D020411:Oligonucleotide Array Sequence Analysis / Q000191:economics / Q000379:methods*; D015345:Oligonucleotide Probes / Q000737:chemistry; D013050:Spectrometry, Fluorescence / Q000191:economics / Q000379:methods; D013997:Time Factors
chemical_list: D005456:Fluorescent Dyes; D035683:MicroRNAs; D015345:Oligonucleotide Probes
keywords: Amplification-free detection; MicroRNA; Microarray; Sandwich Assay