Accurate quantification of 3′-terminal 2′-O-methylated small RNAs by utilizing oxidative deep sequencing and stem-loop RT-qPCR
Yan Kong1, Huanhuan Hu1, Yangyang Shan1, Zhen Zhou1, Ke Zen1, Yulu Sun3, Rong Yang2(), Zheng Fu1(), Xi Chen1,4()
1. Nanjing Drum Tower Hospital Center of Molecular Diagnostic and Therapy, State Key Laboratory of Pharmaceutical Biotechnology, Jiangsu Engineering Research Center for MicroRNA Biology and Biotechnology, NJU Advanced Institute of Life Sciences (NAILS), Institute of Artificial Intelligence Biomedicine, School of Life Sciences, Nanjing University, Nanjing 210023, China 2. Department of Urology, Drum Tower Hospital, Medical School of Nanjing University, Institute of Urology, Nanjing University, Nanjing 210008, China 3. Department of General Surgery, Drum Tower Hospital, Medical school of Nanjing University, Nanjing 210008, China 4. Research Unit of Extracellular RNA, Chinese Academy of Medical Sciences, Nanjing 210023, China
The continuing discoveries of novel classes of RNA modifications in various organisms have raised the need for improving sensitive, convenient, and reliable methods for quantifying RNA modifications. In particular, a subset of small RNAs, including microRNAs (miRNAs) and Piwi-interacting RNAs (piRNAs), are modified at their 3′-terminal nucleotides via 2′-O-methylation. However, quantifying the levels of these small RNAs is difficult because 2′-O-methylation at the RNA 3′-terminus inhibits the activity of polyadenylate polymerase and T4 RNA ligase. These two enzymes are indispensable for RNA labeling or ligation in conventional miRNA quantification assays. In this study, we profiled 3′-terminal 2′-O-methyl plant miRNAs in the livers of rice-fed mice by oxidative deep sequencing and detected increasing amounts of plant miRNAs with prolonged oxidation treatment. We further compared the efficiency of stem-loop and poly(A)-tailed RT-qPCR in quantifying plant miRNAs in animal tissues and identified stem-loop RT-qPCR as the only suitable approach. Likewise, stem-loop RT-qPCR was superior to poly(A)-tailed RT-qPCR in quantifying 3′-terminal 2′-O-methyl piRNAs in human seminal plasma. In summary, this study established a standard procedure for quantifying the levels of 3′-terminal 2′-O-methyl miRNAs in plants and piRNAs. Accurate measurement of the 3′-terminal 2′-O-methylation of small RNAs has profound implications for understanding their pathophysiologic roles in biological systems.
Corresponding Author(s):
Rong Yang,Zheng Fu,Xi Chen
作者简介:
Peng Lu, Renxing Wang, and Yue Xing contributed equally to this work.
引用本文:
. [J]. Frontiers of Medicine, 2022, 16(2): 240-250.
Yan Kong, Huanhuan Hu, Yangyang Shan, Zhen Zhou, Ke Zen, Yulu Sun, Rong Yang, Zheng Fu, Xi Chen. Accurate quantification of 3′-terminal 2′-O-methylated small RNAs by utilizing oxidative deep sequencing and stem-loop RT-qPCR. Front. Med., 2022, 16(2): 240-250.
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