title: Self-reporting hybridisation assay for miRNA analysis.
abstract: Hybridisation assays, which are commonly used to analyse oligonucleotides such as siRNAs and miRNAs, often employ detection probes with fluorescent tags. The signal emitted by a fluorescent tag covers a broad range of wavelengths and this limits the multiplexing potential due to overlapping signals. A novel method of indirect oligonucleotide analysis has been developed which combines a hybridisation assay with cleavable small molecule mass tags using HPLC-ESI MS detection. A self-reporting detection probe has been designed which incorporates a DNA/RNA chimeric oligonucleotide sequence in the reporter region, which generates small nucleotide products upon RNase cleavage of the ribose-phosphate backbone. These small nucleotides can then serve as mass tags for the indirect detection of oligonucleotide analytes. The narrow mass range covered by a small molecule mass tag combined with the wide range of possible mass tags provides a high degree of multiplexing potential. This approach has been demonstrated for the analysis of a synthetic miRNA.
authors.affiliation: Chemistry, FNES, University of Southampton, Southampton, SO17 1BJ, UK. gjl@soton.ac.uk.
authors.forename: Jo-Anne
authors.initials: JA
authors.lastname: Riley
authors.forename: Tom
authors.initials: T
authors.lastname: Brown
authors.forename: Nittaya
authors.initials: N
authors.lastname: Gale
authors.forename: Julie
authors.initials: J
authors.lastname: Herniman
authors.forename: G John
authors.initials: GJ
authors.lastname: Langley
mesh_terms: D000818:Animals; D002417:Cattle; D035683:MicroRNAs / Q000032:analysis* / Q000235:genetics; D009693:Nucleic Acid Hybridization / Q000379:methods*; D021241:Spectrometry, Mass, Electrospray Ionization / Q000379:methods*; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization / Q000379:methods*
chemical_list: D035683:MicroRNAs