Self-reporting hybridisation assay for miRNA analysis

• Published in: Analyst (London) 2014-03, Vol.139 (5), p.188-192
• Main Authors: Riley, Jo-Anne, Brown, Tom, Gale, Nittaya, Herniman, Julie, Langley, G. John
• Format: Article
• Language: English
• Subjects: Animals; Cattle; MicroRNAs - analysis; MicroRNAs - genetics; Nucleic Acid Hybridization - methods; Spectrometry, Mass, Electrospray Ionization - methods; Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization - methods
• ISSN: 0003-2654; 1364-5528
• DOI: 10.1039/c3an01825c

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. Self-reporting probes have been used in a sandwich hybridisation assay for the indirect analysis of miRNA with HPLC-ESI MS detection.