title: Simultaneous Determination of Multiple microRNA Levels Utilizing Biotinylated Dideoxynucleotides and Mass Spectrometry.
abstract: MicroRNAs (miRNAs) are important regulators of gene translation and have been suggested as potent biomarkers in various disease states. In this study, we established an efficient method for simultaneous determination of multiple miRNA levels, employing the previously developed SPC-SBE (solid phase capture-single base extension) approach and MALDI-TOF mass spectrometry (MS). In this approach, we first perform reverse transcription of miRNAs extracted using stem-loop primers. Then the cDNA is co-amplified with competitors, synthetic oligonucleotides whose sequences precisely match cDNA except for one base, and the amplicons serve as templates for a multiplexed SBE reaction. Extension products are isolated using SPC and quantitatively analyzed with MALDI-TOF MS to determine multiple miRNA levels. Here we demonstrated concurrent analysis of four miRNA levels utilizing the approach. Furthermore, we showed the presented method significantly facilitated MS analysis of peak area ratio owing to SPC. The SPC process allowed effective removal of irrelevant reaction components prior to MS and promoted MS sample purification. Data obtained in this study was verified with RT-qPCR and agreement was shown on one order of magnitude scale, suggesting the SPC-SBE and MS approach has strong potential as a viable tool for high throughput miRNA analysis.
authors.affiliation: Graduate School for Biomedical Science & Engineering, Hanyang University, Seoul, Korea.
authors.forename: Sobin
authors.initials: S
authors.lastname: Kim
authors.affiliation: Graduate School for Biomedical Science & Engineering, Hanyang University, Seoul, Korea.
authors.forename: Jungyun
authors.initials: J
authors.lastname: Park
authors.affiliation: School of Interdisciplinary Bioscience and Bioengineering, Pohang University of Science and Technology, Pohang, Gyeongbuk, Korea.
authors.forename: Jeongkyeong
authors.initials: J
authors.lastname: Na
authors.affiliation: School of Interdisciplinary Bioscience and Bioengineering, Pohang University of Science and Technology, Pohang, Gyeongbuk, Korea.|Department of Chemical Engineering, Pohang University of Sciences and Technology, Pohang, Gyeongbuk, Korea.
authors.forename: Gyoo Yeol
authors.initials: GY
authors.lastname: Jung
authors.affiliation: Graduate School for Biomedical Science & Engineering, Hanyang University, Seoul, Korea.|Department of Medical Genetics, College of Medicine, Hanyang University, Seoul, Korea.
authors.forename: Jungwook
authors.initials: J
authors.lastname: Hwang
mesh_terms: D000072283:A549 Cells; D000818:Animals; D019921:Biotinylation; D002138:Calibration; D018076:DNA, Complementary / Q000737:chemistry / Q000235:genetics; D054306:Dideoxynucleotides / Q000235:genetics* / Q000378:metabolism; D059014:High-Throughput Nucleotide Sequencing / Q000379:methods; D006801:Humans; D035683:MicroRNAs / Q000235:genetics* / Q000378:metabolism; D015203:Reproducibility of Results; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization / Q000379:methods*
chemical_list: D018076:DNA, Complementary; D054306:Dideoxynucleotides; C555159:MIRN142 microRNA, human; C534612:MIRN31 microRNA, human; C000609796:MIRN487 microRNA, mouse; D035683:MicroRNAs