摘要
建立了高效液相色谱法(HPLC)同时检测细胞基因组中5种去甲基化相关修饰核苷含量的方法。以易位蛋白TET1过表达HEK293T细胞系(HEK293T(TET1CD))为研究对象,采用DNA Dot Blot法和HPLC法验证过表达TET1细胞中修饰核苷发生的变化。9种核苷在120 min内实现完全分离,且线性关系良好(R2>0.995),检出限为0.1068~0.5278μmol/L,定量限为0.3236~1.5995μmol/L。HEK293T(TET1CD)细胞经DNA Dot Blot法检测,诱导后细胞基因组中5-羟甲基脱氧胞嘧啶(5hmdC)含量显著升高(P<0.0001);HPLC检测显示,诱导细胞基因组中5hmdC和5-羟甲基脱氧尿嘧啶(5hmdU)含量升高(P<0.05),5-甲基脱氧胞嘧啶(5mdC)含量下降(P<0.05),5-甲酰基脱氧胞嘧啶(5fdC)和5-羧基脱氧胞嘧啶(5cadC)未检出。检出样本回收率在100.2%~113.5%之间,相对标准偏差在0.72%~3.0%之间。本研究建立的HPLC方法,能够同时精确检测基因组中5种修饰核苷的含量,能够替代传统的DNA Dot Blot法,为研究去甲基化中的碱基代谢产物提供灵敏、可靠的检测途径。
A high-performance liquid chromatography(HPLC)method was established for the simultaneous detection of five demethylation-associated modified nucleoside contents in cell genomes.The changes of the modified nucleosides in the overexpressed TET1 cell line(HEK293T(TET1CD))were verified by DNA Dot Blot and HPLC using the translocator protein HEK293T(TET1CD).Nine nucleosides were completely separated within 120 min,and the linear relationship was good(R2>0.995),with the limits of detection ranging from 0.1068 to 0.5278μmol/L,and the limits of quantification in the range of 0.3236‒1.5995μmol/L.HEK293T(TET1CD)cells showed that the content of 5-hydroxymethyl-2'-deoxycytidine(5hmdC)in the genome of the induced cells was significantly elevated by the DNA Dot Blot assay(P<0.0001);HPLC assay showed that the 5hmdC and 5-hydroxymethyl-2'-deoxyuridine(5hmdU)contents increased(P<0.05),the 5-methyl-2'-deoxycytidine(5mdC)content decreased(P<0.05),and 5-formyl-2'-deoxycytidine(5fdC)and 5-carboxyl-2'-deoxycytidine(5cadC)were not detected in the genome of induced cells.The recoveries of the spiked samples ranged from 100.2%to 113.5%,with the relative standard deviations of 0.72%‒3.0%.The established HPLC method can simultaneously and accurately detect the contents of the five modified nucleosides in the genome,replacing the traditional DNA Dot Blot method to provide a sensitive and reliable detection route for the study of base metabolites in demethylation.
作者
杨喜善
莫俊
罗庆元
周艳华
张鹏
曾柱
黄文竹
YANG Xishan;MO Jun;LUO Qingyuan;ZHOU Yanhua;ZHANG Peng;ZENG Zhu;HUANG Wenzhu(School of Biology&Engineering,Guizhou Medical University,Guiyang 550004,China;Center for Tissue Engineering and Stem Cell Research,Key Laboratory of Adult Stem Cell Transformation Research,Chinese Academy of Medical Sciences,Guiyang 550004,China)
出处
《分析试验室》
北大核心
2025年第7期999-1005,共7页
Chinese Journal of Analysis Laboratory
基金
国家自然科学基金(81960036)
贵州省科技计划项目(黔科合基础-ZK[2021]重点005)资助。
