Attomole quantification and global profile of RNA modifications: Epitranscriptome of human neural stem cells.
Attomole quantification and global profile of RNA modifications: Epitranscriptome of human neural stem cells.
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DOI：
10.1093/nar/gkv971
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发表时间：
2016-02-18
影响因子：
14.9
通讯作者：
Agris PF
中科院分区：
文献类型：
--
作者：
Basanta-Sanchez M;Temple S;Ansari SA;D'Amico A;Agris PF
探索表转录组需要开发高度灵敏和准确的技术，以阐明100多种RNA修饰对细胞过程的贡献。一个高度灵敏和准确的超高效液相色谱-串联质谱法的开发，同时检测和定量28个修改和四个主要核苷在不到20分钟。绝对浓度计算使用消光系数的每一个研究的RNA修饰。UV图谱和消光系数的全面RNA修饰数据库的相对标准偏差在2.3- 5.2%范围内。在0.99227-0.99999范围内观察到良好的线性，检测限值范围为63.75阿托摩尔至1.21飞摩尔。通过分析来自100 ng人多能干细胞衍生的神经细胞的RNA的RNA修饰来评价分析性能。在比相应的亲本核苷低四个数量级的浓度下检测到修饰，并且低至23.01毫微微克，64.09阿托摩尔。RNA修饰的直接和全局定量分析是这种新方法的优点之一。
Exploration of the epitranscriptome requires the development of highly sensitive and accurate technologies in order to elucidate the contributions of the more than 100 RNA modifications to cell processes. A highly sensitive and accurate ultra-high performance liquid chromatography—tandem mass spectrometry method was developed to simultaneously detect and quantify 28 modified and four major nucleosides in less than 20 min. Absolute concentrations were calculated using extinction coefficients of each of the RNA modifications studied. A comprehensive RNA modifications database of UV profiles and extinction coefficient is reported within a 2.3–5.2 % relative standard deviation. Excellent linearity was observed 0.99227–0.99999 and limit of detection values ranged from 63.75 attomoles to 1.21 femtomoles. The analytical performance was evaluated by analyzing RNA modifications from 100 ng of RNA from human pluripotent stem cell-derived neural cells. Modifications were detected at concentrations four orders of magnitude lower than the corresponding parental nucleosides, and as low as 23.01 femtograms, 64.09 attomoles. Direct and global quantitative analysis of RNA modifications are among the advantages of this new approach.