Dot Blot Analysis for Measuring Global N⁶-Methyladenosine Modification of RNA

Posttranscriptional modification of mRNAs plays an important role in establishing the functional diversity of the proteome. The m⁶A modification is found in many species of RNA, including tRNA, mRNA, rRNA, and long noncoding RNAs. The physiological role of m⁶A modification of RNA is not fully explored and is a topic of current research. It is predicted that the major effect of m⁶A modification of mRNAs is on its stability and/or translation. The global changes in m⁶A levels in total RNA or particular species of RNAs can be measured by dot blot analysis using m⁶A specific antibodies or using mass spectrometry following chromatographic separation. The dot blot method for detection of global m⁶A changes is a relatively straightforward method to quantitate m⁶A modification but suffers from low sensitivity when the fraction of m⁶A-modified RNA is small in analyzed samples. Here, we describe a modified dot blot method that is sensitive and quantitative for detecting m⁶A-modified RNA by adding an immunoprecipitation step to enrich for m⁶A-modified RNA.