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

作     者：Yan Kong Huanhuan Hu Yangyang Shan Zhen Zhou Ke Zen Yulu Sun Rong Yang Zheng Fu Xi Chen Yan Kong;Huanhuan Hu;Yangyang Shan;Zhen Zhou;Ke Zen;Yulu Sun;Rong Yang;Zheng Fu;Xi Chen

作者机构：Nanjing Drum Tower Hospital Center of Molecular Diagnostic and TherapyState Key Laboratory of Pharmaceutical BiotechnologyJiangsu Engineering Research Center for MicroRNA Biology and BiotechnologyNJU Advanced Institute of Life Sciences(NAILS)Institute of Artificial Intelligence BiomedicineSchool of Life SciencesNanjing UniversityNanjing 210023China Department of UrologyDrum Tower HospitalMedical School of Nanjing UniversityInstitute of UrologyNanjing UniversityNanjing 210008China Department of General SurgeryDrum Tower HospitalMedical school of Nanjing UniversityNanjing 210008China Research Unit of Extracellular RNAChinese Academy of Medical SciencesNanjing 210023China 

出 版 物：《Frontiers of Medicine》 (医学前沿（英文版）)

年 卷 期：2022年第16卷第2期

页      面：240-250页

核心收录：

学科分类：0831[工学-生物医学工程（可授工学、理学、医学学位）] 1002[医学-临床医学] 08[工学] 0836[工学-生物工程] 

基　　金：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) Research Unit of Extracellular Non-Coding RNA,Chinese Academy of Medical Sciences(No.2021RU015) 

主　　题：small RNAs 2'-0-methylation sequencing RT-qPCR 

摘      要：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 *** particular,a subset of small RNAs,including microRNAs(miRNAs)and Piwi-interacting RNAs(piRNAs),are modified at their 3 -terminal nucleotides via 2 -***,quantifying the levels of these small RNAs is difficult because 2 -0-methylation at the RNA 3 -terminus inhibits the activity of polyadenylate polymerase and T4 RNA *** two enzymes are indispensable for RNA labeling or ligation in conventional miRNA quantification *** this study,we profiled 3 -terminal 2 -0-methyl plant miRNAs in the livers of rice-fed mice by oxidative deep sequencing and detected increasing amounts of plant miRNAs with prolonged oxidation *** 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 ***,stem-loop RT-qPCR was superior to poly(A)-tailed RT-qPCR in quantifying 3 -terminal 2 -0-methyl piRNAs in human seminal *** summary,this study established a standard procedure for quantifying the levels of 3 -terminal 2 -0-methyl miRNAs in plants and *** measurement of the 3 -terminal 2 -0-methylation of small RNAs has profound implications for understanding their pathophysiologic roles in biological systems.