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Accurate quantification of 3′-terminal 2′-O-methylated small RNAs by utilizing oxidative deep sequencing and stem-loop RT-qPCR

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Abstract

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.

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Acknowledgements

This work was supported by the Fundamental Research Funds for the Central Universities (No. 020814380146), National Basic Research Program of China (973 Program) (No. 2014CB542300), National Natural Science Foundation of China (Nos. 32022015, 32001077, 31871295, 21877060, 81250044, 81602697, and 81772727), and Research Unit of Extracellular Non-Coding RNA, Chinese Academy of Medical Sciences (No. 2021RU015).

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Authors and Affiliations

  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

    Yan Kong, Huanhuan Hu, Yangyang Shan, Zhen Zhou, Ke Zen, Zheng Fu & Xi Chen

  2. Department of Urology, Drum Tower Hospital, Medical School of Nanjing University, Institute of Urology, Nanjing University, Nanjing, 210008, China

    Rong Yang

  3. Department of General Surgery, Drum Tower Hospital, Medical school of Nanjing University, Nanjing, 210008, China

    Yulu Sun

  4. Research Unit of Extracellular RNA, Chinese Academy of Medical Sciences, Nanjing, 210023, China

    Xi Chen

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  1. Yan Kong
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  2. Huanhuan Hu
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  3. Yangyang Shan
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  4. Zhen Zhou
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  5. Ke Zen
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  6. Yulu Sun
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  7. Rong Yang
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  8. Zheng Fu
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  9. Xi Chen
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Correspondence to Rong Yang, Zheng Fu or Xi Chen.

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Compliance with ethics guidelines

Yan Kong, Huanhuan Hu, Yangyang Shan, Zhen Zhou, Ke Zen, Yulu Sun, Rong Yang, Zheng Fu, and Xi Chen declare that they have no conflict of interest. The study protocol was approved by the Institutional Review Board of Nanjing University (Nanjing, China) (Approval No. IACUC-2006008). All procedures followed were in accordance with the ethical standards of the responsible committee on human experimentation (institutional and national) and with the Helsinki Declaration of 1975, as revised in 2000 (5). Informed consent was obtained from all patients who provided semen samples.

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Kong, Y., Hu, H., Shan, Y. et al. Accurate quantification of 3′-terminal 2′-O-methylated small RNAs by utilizing oxidative deep sequencing and stem-loop RT-qPCR. Front. Med. 16, 240–250 (2022). https://doi.org/10.1007/s11684-021-0909-7

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