Dot Blot Analysis of N6-methyladenosine RNA Modification Levels
N6-甲基腺苷RNA修饰水平的斑点印迹分析
发布: 2017年01月05日第7卷第1期 DOI: 10.21769/BioProtoc.2095 浏览次数: 29836
评审: Antoine de MorreeXiaoyi ZhengVaibhav B Shah
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参见作者原研究论文
The authors used this protocol in:
Jul 2016
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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 (斑点印迹)
RNA modification (RNA修饰)
m6A (N6-甲基腺苷)
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
- Amersham Hybond-N+ membrane (GE Healthcare, catalog number: RPN203B )
- Plastic wrap
- Amersham Hyperfilm ECL (GE Healthcare, catalog number: 28906835 )
- Total RNA
- Dynabeads® mRNA Purification Kit (Thermo Fisher Scientific, AmbionTM, catalog number: 61006 )
- RNase-free water
- Anti-m6A antibody (Synaptic Systems, catalog number: 202 003 )
- Goat anti-rabbit IgG-HRP (Santa Cruz Biotechnology, catalog number: sc-2004 )
- ECL Western Blotting Substrate (Thermo Fisher Scientific, Thermo ScientificTM, catalog number: 32106 )
- 1x phosphate buffered saline (1x PBS), pH 7.4
- Tween 20 (Sigma-Aldrich, catalog number: P9416 )
- Non-fat milk (Bio-Rad Laboratories, catalog number: 1706404 )
- Wash buffer (see Recipes)
- Blocking buffer (see Recipes)
- Antibody dilution buffer (see Recipes)
Equipment
- NanoDrop 2000 spectrophotometer (Thermo Fisher Scientific, Thermo ScientificTM, model: NanoDropTM 2000 Spectrophotometer )
- Heat block
- Stratalinker 2400 UV Crosslinker (Stratalinker)
- Shaker
Software
- ImageJ
Procedure
文章信息
版权信息
© 2017 The Authors; exclusive licensee Bio-protocol LLC.
如何引用
Shen, L., Liang, Z. and Yu, H. (2017). Dot Blot Analysis of N6-methyladenosine RNA Modification Levels. Bio-protocol 7(1): e2095. DOI: 10.21769/BioProtoc.2095.
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分类
植物科学 > 植物生物化学 > RNA > RNA 提取
分子生物学 > RNA > RNA 检测
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