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Modification of nucleotides significantly increases the diversity of functional nucleic acids. As one of the most common modifications of RNAs, methylation of the 2′-hydroxyl-group of ribonucleotides (2′- O -methylation) has been found in various RNAs in eukaryotes. However, due to the lack of an efficient method for quantifying small RNA 3′ terminal 2′- O -methylation, it is difficult to monitor the dynamic change of 3′ terminal 2′- O -methylation during various biological processes. Capitalizing on the finding that 3′ terminal RNA 2′- O -methylation can inhibit the activity of poly(A) polymerase, an enzyme that can add the poly(A)-tail to RNA, we develop a method by which the 2′- O -methylation level of small RNAs, such as microRNAs (miRNAs) and Piwi-interacting RNAs (piRNAs), can be directly quantified based on the poly(A)-tailed RT-qPCR technique. With this method, we successfully determine the 2′- O -methylation level of miRNAs in  Arabidopsis thaliana  and mouse lung tissue, piRNA in human seminal plasma, and monitor the alteration of miRNA 2′- O -methylation in  Drosophila  Schneider 2 cells after knockdown of  Drosophila  methyltransferase protein Hua enhancer 1 (DmHen-1).

Direct quantification of 3′ terminal 2′-O-methylation of small RNAs by RT-qPCR