Attomole quantification and global profile of RNA modifications: Epitranscriptome of human neural stem cells

Maria Basanta-Sanchez, Sally Temple, Suraiya A. Ansari, Anna D'Amico, Paul F. Agris

Research output: Contribution to journalArticlepeer-review

76 Citations (Scopus)

Abstract

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 from63.75 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.

Original languageEnglish
Article numbere26
JournalNucleic acids research
Volume44
Issue number3
DOIs
Publication statusPublished - Feb 18 2016
Externally publishedYes

ASJC Scopus subject areas

  • Genetics

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