A Method for Measuring RNA N6-methyladenosine Modifications in Cells and Tissues

• Published in: Journal of visualized experiments no. 118
• Main Authors: Wang, Chao-Yung, Lin, Mei-Hsiu, Su, Hui-Ting
• Format: Journal Article
• Language: English
• Published: MyJove Corporation 05.12.2016
• Subjects: Biochemistry
• ISSN: 1940-087X; 1940-087X
• DOI: 10.3791/54672

N 6 -Methyladenosine (m 6 A) modifications of RNA are diverse and ubiquitous amongst eukaryotes. They occur in mRNA, rRNA, tRNA, and microRNA. Recent studies have revealed that these reversible RNA modifications affect RNA splicing, translation, degradation, and localization. Multiple physiological processes, like circadian rhythms, stem cell pluripotency, fibrosis, triglyceride metabolism, and obesity are also controlled by m 6 A modifications. Immunoprecipitation/sequencing, mass spectrometry, and modified northern blotting are some of the methods commonly employed to measure m 6 A modifications. Herein, we present a northeastern blotting technique for measuring m 6 A modifications. The current protocol provides good size separation of RNA, better accommodation and standardization for various experimental designs, and clear delineation of m 6 A modifications in various sources of RNA. While m 6 A modifications are known to have a crucial impact on human physiology relating to circadian rhythms and obesity, their roles in other (patho)physiological states are unclear. Therefore, investigations on m 6 A modifications have immense possibility to provide key insights into molecular physiology.