Fig. 2.

Detection of methylated bases in mature miRNAs. a Schematic depiction of the procedure for detecting RNA modifications in mature miRNA sequences. Total small RNA extracted from cells was hybridized with oligonucleotides complementary to target miRNAs on magnetic beads. Captured miRNAs were eluted and applied to sample plates and then analyzed by matrix-assisted laser desorption/ionization time-of-flight tandem MS (MALDI-TOF-MS/MS). b Dynamic range of methylated miRNA detection. Synthetic miR-200c-3p oligonucleotides with or without methylation were mixed at the indicated concentrations and analyzed by MALDI-TOF-MS/MS. c Mass spectrum of miR-17-5p and let-7a-5p obtained from pancreatic cancer patient-derived tissue. The spectrum shows monovalent, divalent, and trivalent methylated miR-17-5p RNA peaks and monovalent and divalent let-7a-5p RNA peaks (see Methods for details). d Position of methylated nucleoside in each miRNA