title: Immuno-Northern Blotting: Detection of RNA Modifications by Using Antibodies against Modified Nucleosides.
abstract: The biological roles of RNA modifications are still largely not understood. Thus, developing a method for detecting RNA modifications is important for further clarification. We developed a method for detecting RNA modifications called immuno-northern blotting (INB) analysis and herein introduce its various capabilities. This method involves the separation of RNAs using either polyacrylamide or agarose gel electrophoresis, followed by transfer onto a nylon membrane and subsequent immunoblotting using antibodies against modified nucleosides for the detection of specific modifications. We confirmed that INB with the antibodies for 1-methyladenosine (m1A), N6-methyladenosine (m6A), pseudouridine, and 5-methylcytidine (m5C) showed different modifications in a variety of RNAs from various species and organelles. INB with the anti-m5C antibody revealed that the antibody cross-reacted with another modification on DNA, suggesting the application of this method for characterization of the antibody for modified nucleosides. Additionally, using INB with the antibody for m1A, which is a highly specific modification in eukaryotic tRNA, we detected tRNA-derived fragments known as tiRNAs under the cellular stress response, suggesting the application for tracking target RNA containing specific modifications. INB with the anti-m6A antibody confirmed the demethylation of m6A by the specific demethylases fat mass and obesity-associated protein (FTO) and ALKBH5, suggesting its application for quantifying target modifications in separated RNAs. Furthermore, INB demonstrated that the knockdown of FTO and ALKBH5 increased the m6A modification in small RNAs as well as in mRNA. The INB method has high specificity, sensitivity, and quantitative capability, and it can be employed with conventional experimental apparatus. Therefore, this method would be useful for research on RNA modifications and metabolism.
authors.affiliation: Division of Nephrology, Endocrinology, and Vascular Medicine, Tohoku University Graduate School of Medicine, Sendai, Japan.|Tohoku Medical Megabank Organization, Sendai, Japan.
authors.forename: Eikan
authors.initials: E
authors.lastname: Mishima
authors.affiliation: Laboratory of Oncology, Pharmacy Practice and Sciences, Tohoku University Graduate School of Pharmaceutical Sciences, Sendai, Japan.
authors.forename: Daisuke
authors.initials: D
authors.lastname: Jinno
authors.affiliation: Division of Nephrology, Endocrinology, and Vascular Medicine, Tohoku University Graduate School of Medicine, Sendai, Japan.|Tohoku Medical Megabank Organization, Sendai, Japan.
authors.forename: Yasutoshi
authors.initials: Y
authors.lastname: Akiyama
authors.affiliation: Department of Clinical Pharmacology and Genetics, School of Pharmaceutical Sciences, University of Shizuoka, Shizuoka, Japan.
authors.forename: Kunihiko
authors.initials: K
authors.lastname: Itoh
authors.affiliation: Laboratory of Oncology, Pharmacy Practice and Sciences, Tohoku University Graduate School of Pharmaceutical Sciences, Sendai, Japan.
authors.forename: Shinnosuke
authors.initials: S
authors.lastname: Nankumo
authors.affiliation: Division of Nephrology, Endocrinology, and Vascular Medicine, Tohoku University Graduate School of Medicine, Sendai, Japan.
authors.forename: Hisato
authors.initials: H
authors.lastname: Shima
authors.affiliation: Division of Nephrology, Endocrinology, and Vascular Medicine, Tohoku University Graduate School of Medicine, Sendai, Japan.
authors.forename: Koichi
authors.initials: K
authors.lastname: Kikuchi
authors.affiliation: Division of Nephrology, Endocrinology, and Vascular Medicine, Tohoku University Graduate School of Medicine, Sendai, Japan.
authors.forename: Yoichi
authors.initials: Y
authors.lastname: Takeuchi
authors.affiliation: Department of Neurosurgery, Tohoku University Graduate School of Medicine, Sendai, Japan.
authors.forename: Alaa
authors.initials: A
authors.lastname: Elkordy
authors.affiliation: Division of Nephrology, Endocrinology, and Vascular Medicine, Tohoku University Graduate School of Medicine, Sendai, Japan.
authors.forename: Takehiro
authors.initials: T
authors.lastname: Suzuki
authors.affiliation: Department of Neurosurgery, Tohoku University Graduate School of Medicine, Sendai, Japan.
authors.forename: Kuniyasu
authors.initials: K
authors.lastname: Niizuma
authors.affiliation: Division of Nephrology, Endocrinology, and Vascular Medicine, Tohoku University Graduate School of Medicine, Sendai, Japan.
authors.forename: Sadayoshi
authors.initials: S
authors.lastname: Ito
authors.affiliation: Laboratory of Oncology, Pharmacy Practice and Sciences, Tohoku University Graduate School of Pharmaceutical Sciences, Sendai, Japan.
authors.forename: Yoshihisa
authors.initials: Y
authors.lastname: Tomioka
authors.affiliation: Division of Nephrology, Endocrinology, and Vascular Medicine, Tohoku University Graduate School of Medicine, Sendai, Japan.|Department of Medical Science, Tohoku University Graduate School of Biomedical Engineering, Sendai, Japan.|Department of Clinical Biology and Hormonal Regulation, Tohoku University Graduate School of Medicine, Sendai, Japan.
authors.forename: Takaaki
authors.initials: T
authors.lastname: Abe
mesh_terms: D000911:Antibodies, Monoclonal / Q000276:immunology; D015152:Blotting, Northern / Q000379:methods*; D009705:Nucleosides / Q000737:chemistry; D012313:RNA / Q000737:chemistry / Q000276:immunology; D012680:Sensitivity and Specificity
chemical_list: D000911:Antibodies, Monoclonal; D009705:Nucleosides; D012313:RNA