Dot Blot Analysis for Measuring Global N6-Methyladenosine Modification of RNA
Dot Blot Analysis for Measuring Global N6-Methyladenosine Modification of RNA
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DOI：
10.1007/978-1-4939-8808-2_20
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发表时间：
2019-01-01
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影响因子：
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通讯作者：
Wajapeyee, Narendra
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文献类型：
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作者：
Nagarajan, Arvindhan;Janostiak, Radoslav;Wajapeyee, Narendra
mRNA的转录后修饰在建立蛋白质组的功能多样性中起着重要作用。m（6）A修饰存在于许多种类的RNA中，包括tRNA、mRNA、rRNA和长链非编码RNA。m（6）A修饰RNA的生理作用尚未完全研究，是当前研究的一个主题。据预测，m（6）A修饰的mRNA的主要影响是其稳定性和/或翻译。总RNA或特定RNA种类中m（6）A水平的总体变化可以通过使用m（6）A特异性抗体的斑点印迹分析或使用色谱分离后的质谱法来测量。用于检测整体m（6）A变化的斑点印迹法是定量m（6）A修饰的相对直接的方法，但是当分析样品中m（6）A修饰的RNA的分数小时，灵敏度低。在这里，我们描述了一种改良的斑点印迹法，通过增加免疫沉淀步骤富集m（6）A修饰的RNA，该方法灵敏且定量地检测m（6）A修饰的RNA。
Posttranscriptional modification of mRNAs plays an important role in establishing the functional diversity of the proteome. The m(6)A modification is found in many species of RNA, including tRNA, mRNA, rRNA, and long noncoding RNAs. The physiological role of m(6)A modification of RNA is not fully explored and is a topic of current research. It is predicted that the major effect of m(6)A modification of mRNAs is on its stability and/or translation. The global changes in m(6)A levels in total RNA or particular species of RNAs can be measured by dot blot analysis using m(6)A specific antibodies or using mass spectrometry following chromatographic separation. The dot blot method for detection of global m(6)A changes is a relatively straightforward method to quantitate m(6)A modification but suffers from low sensitivity when the fraction of m(6)A-modified RNA is small in analyzed samples. Here, we describe a modified dot blot method that is sensitive and quantitative for detecting m(6)A-modified RNA by adding an immunoprecipitation step to enrich for m(6)A-modified RNA.