title: A Method for Measuring RNA N 6-methyladenosine Modifications in Cells and Tissues.
abstract: N6-Methyladenosine (m6A) 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 m6A modifications. Immunoprecipitation/sequencing, mass spectrometry, and modified northern blotting are some of the methods commonly employed to measure m6A modifications. Herein, we present a northeastern blotting technique for measuring m6A modifications. The current protocol provides good size separation of RNA, better accommodation and standardization for various experimental designs, and clear delineation of m6A modifications in various sources of RNA. While m6A 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 m6A modifications have immense possibility to provide key insights into molecular physiology.
authors.affiliation: Department of Cardiology, Chang Gung Memorial Hospital, Chang Gung University College of Medicine; cwang@ocean.ag.
authors.forename: Chao-Yung
authors.initials: CY
authors.lastname: Wang
authors.affiliation: Department of Cardiology, Chang Gung Memorial Hospital, Chang Gung University College of Medicine.
authors.forename: Mei-Hsiu
authors.initials: MH
authors.lastname: Lin
authors.affiliation: Department of Cardiology, Chang Gung Memorial Hospital, Chang Gung University College of Medicine.
authors.forename: Hui-Ting
authors.initials: HT
authors.lastname: Su
mesh_terms: D000241:Adenosine / Q000031:analogs & derivatives* / Q000737:chemistry; D015152:Blotting, Northern / Q000379:methods*; D006801:Humans; D008745:Methylation; D035683:MicroRNAs; D012313:RNA / Q000737:chemistry*; D012333:RNA, Messenger; D012335:RNA, Ribosomal; D012343:RNA, Transfer
chemical_list: D035683:MicroRNAs; D012333:RNA, Messenger; D012335:RNA, Ribosomal; D012313:RNA; D012343:RNA, Transfer; C010223:N-methyladenosine; D000241:Adenosine