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.
Highlights
MicroRNA, a class of small (18-23nt) noncoding RNA, regulates translation of gene transcripts by typically suppressing the expression of target mRNAs [1,2]
For quantitative mass spectrometry (MS) analysis of miRNA levels, we established an external calibration curve using two synthetic oligonucleotide templates (Fig 2). Both C- and G- extension peaks with good signal intensity were observed for all template ratios tested (C/G ratio of 0.5, 1, 2, 5, 10 and 20)
Template ratio was not linearly proportional to the MS peak area ratio. This could be due to the difference in the extension reaction rates of biotin-ddCTP and biotin-ddGTP or in ionization efficiencies during MS analysis between C and G extension products
Summary
MicroRNA (miRNA), a class of small (18-23nt) noncoding RNA, regulates translation of gene transcripts by typically suppressing the expression of target mRNAs [1,2]. A large number of studies investigating roles of miRNA in the alteration of gene translation have been conducted to characterize disease states and to identify therapeutic pathways [8,9,10]. Most of these studies involve quantification of a group of miRNAs. Conventional gold standards of PLOS ONE | DOI:10.1371/journal.pone.0153201. Conventional gold standards of PLOS ONE | DOI:10.1371/journal.pone.0153201 July 5, 2016
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