Abstract
N6-methyladenosine (m6A) is the most prevalent internal modification of eukaryotic messenger RNA (mRNA). The total amount of m6A can be detected by several methods, such as dot blot analysis using specific m6A antibodies and quantitative liquid chromatography-tandem mass spectrometry (LC-MS/MS) (Fu et al., 2014; Shen et al., 2016). Here we describe the method for fast detection of total m6A levels in mRNA by dot blot analysis using a specific m6A antibody.
Keywords: Dot blot, RNA modification, m6A
Background
Dot blot analysis for detecting total m6A levels in mRNA is relatively easy, fast, and cost-effective as compared to other methods, such as two-dimensional thin layer chromatography and LC-MS/MS. This approach can be used, in a qualitative manner, to evaluate temporal and spatial changes in m6A levels in various plant tissues or plants at different developmental stages. This is particularly useful for initial examination of changes in m6A levels in relevant mutants prior to detailed investigations by other complex and quantitative approaches.
Materials and Reagents
Equipment
Software
Procedure
Data analysis
As dot blot analysis is a semi-quantitative approach, the analysis should be repeated through the above procedures using independent biological materials. Only the repeatable changes in m6A levels observed in independent materials as compared to the wild-type control are considered ‘positive’ results, which may be further investigated by other quantitative approaches. In addition, the signals from the dot blot images can be quantified by ImageJ and the statistical analysis should be based on at least three biological replicates.
Representative data
For representative data, please see the paper of Shen et al., 2016.
Notes
This protocol is also applicable to detect other types of RNA modifications if the corresponding specific primary antibodies and secondary antibodies are available.
Recipes
Note: It is unnecessary to use RNase-free water to prepare the above solutions.
Acknowledgments
This work was supported by Academic Research Fund (MOE2015-T2-1-002) from the Ministry of Education-Singapore, the Singapore National Research Foundation Investigatorship Programme (NRF-NRFI2016-02), and the intramural research support from National University of Singapore and Temasek Life Sciences Laboratory.
References
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I have limited experience with this protocol. It seems that the mRNA quantity is very important. 20 ug mRNA perhaps is the lower limit for decent signals. I imagine that you would need 200 ug total RNA to have the same amount of signal. Why don't you try Trizol and let us know the results.
In our experiments, we used RNeasy plus mini kit (QIAGEN) to extract the total RNA. We also tried to this protocol on total RNA, it worked. However, please note that m6A is also found in rRNA, tRNA and snRNA.