Epitranscriptomics - the study of RNA modifications - is an emerging and promising research field. RNA modifications are involved in all steps of post-transcriptional regulation of gene expression (RNA structure, splicing, export; stability, cellular localization and translation). Finally, theses modifications are essential for key cellular functions such as survival, growth and differentiation.
Here we describe an experimental workflow for the quantification of methylated nucleosides from cellular mRNA based on separation and detection on a LCMS-8060 system.
In total, more than 170 RNA modifications - including RNA methylation - have been described. These modifications are present observed on all common RNA types (mRNA, tRNA, rRNA and, ncRNA).
The most common and abundant modifications on RNA in eukaryotes is N6-methyladenosine (m6A). m6A is important for mRNA regulation in physiological condition. As such, any dysregulation of m6A level may lead to disease [1]. This modification is not limited to mRNA but is also observed in other RNA species (rRNA, tRNA, snRNA).
Common technologies to detect m6A modifications [2] use next-generation sequencing techniques . However, these approaches cannot permit multiplex analysis of RNA methylations from a single sample. In addition, they do note enable precise quantification of individual chemical mark.
Mass spectrometry coupled to liquid chromatography has become a method of choice for accurately quantifying modified nucleosides from a biological sample. Here we present an application for the measurement of 14 distinct methylated nucleoside species using LC-MS/MS (Shimadzu LCMS-8060).
>> Download Application Note as a PDF
Find out more on Shimadzu's Website here.
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