Received: AugAccepted: NovemPublished: November 25, 2015Ĭopyright: © 2015 Mishima et al. PLoS ONE 10(11):Įditor: Thomas Preiss, The John Curtin School of Medical Research, AUSTRALIA (2015) Immuno-Northern Blotting: Detection of RNA Modifications by Using Antibodies against Modified Nucleosides. Therefore, this method would be useful for research on RNA modifications and metabolism.Ĭitation: Mishima E, Jinno D, Akiyama Y, Itoh K, Nankumo S, Shima H, et al. The INB method has high specificity, sensitivity, and quantitative capability, and it can be employed with conventional experimental apparatus. Furthermore, INB demonstrated that the knockdown of FTO and ALKBH5 increased the m 6A modification in small RNAs as well as in mRNA. INB with the anti-m 6A antibody confirmed the demethylation of m 6A by the specific demethylases fat mass and obesity-associated protein (FTO) and ALKBH5, suggesting its application for quantifying target modifications in separated RNAs. Additionally, using INB with the antibody for m 1A, 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-m 5C 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. We confirmed that INB with the antibodies for 1-methyladenosine (m 1A), N6-methyladenosine (m 6A), pseudouridine, and 5-methylcytidine (m 5C) showed different modifications in a variety of RNAs from various species and organelles. 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 developed a method for detecting RNA modifications called immuno-northern blotting (INB) analysis and herein introduce its various capabilities. Thus, developing a method for detecting RNA modifications is important for further clarification.

The biological roles of RNA modifications are still largely not understood.

Northern blot analysis