Single-base validation using ligation-amplification method and qPCR

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Fig. 3 Single-base validation using ligation-amplification method and qPCR. Single-base validation using ligation-amplification method and qPCR. (A) Schematic diagram of ligation-amplification method for single-base m6A validation. (B) Validation results of six individual sites. Five of the sites are validated to be m6A sites, whereas the other one is confirmed to be not modified. Data for m6A-CLIP, miCLIP-CITS, miCLIP-CIMS, and MeRIP-seq are downloaded from published literatures. (C) Schematic diagram of qPCR to quantify the methylation level of a specific m6A site. The undigested mRNA sample is treated as control. (D) qPCR results and methylation ratios of six m6A sites. H1 to H3 represent the highly methylated sites (>0.75), while L1 to L3 represent the weakly methylated sites (<0.35). The left y axis represents the ΔΔCt values, and the right y axis represents the methylation ratio determined by m6A-REF-seq. GAPDH, glyceraldehyde-3-phosphate dehydrogenase. Zhang Zhang et al. Sci Adv 2019;5:eaax0250 Copyright © 2019 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works. Distributed under a Creative Commons Attribution NonCommercial License 4.0 (CC BY-NC).