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Dot Blot Analysis of N6-methyladenosine RNA Modification Levels

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Developmental Cell
Jul 2016



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)


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

  1. Amersham Hybond-N+ membrane (GE Healthcare, catalog number: RPN203B )
  2. Plastic wrap
  3. Amersham Hyperfilm ECL (GE Healthcare, catalog number: 28906835 )
  4. Total RNA
  5. Dynabeads® mRNA Purification Kit (Thermo Fisher Scientific, AmbionTM, catalog number: 61006 )
  6. RNase-free water
  7. Anti-m6A antibody (Synaptic Systems, catalog number: 202 003 )
  8. Goat anti-rabbit IgG-HRP (Santa Cruz Biotechnology, catalog number: sc-2004 )
  9. ECL Western Blotting Substrate (Thermo Fisher Scientific, Thermo ScientificTM, catalog number: 32106 )
  10. 1x phosphate buffered saline (1x PBS), pH 7.4
  11. Tween 20 (Sigma-Aldrich, catalog number: P9416 )
  12. Non-fat milk (Bio-Rad Laboratories, catalog number: 1706404 )
  13. Wash buffer (see Recipes)
  14. Blocking buffer (see Recipes)
  15. Antibody dilution buffer (see Recipes)


  1. NanoDrop 2000 spectrophotometer (Thermo Fisher Scientific, Thermo ScientificTM, model: NanoDropTM 2000 Spectrophotometer )
  2. Heat block
  3. Stratalinker 2400 UV Crosslinker (Stratalinker)
  4. Shaker


  1. ImageJ


  1. mRNA purification
    1. Isolate mRNA from total RNA using the Dynabeads® mRNA Purification Kit following the manufacturer’s instructions. For one dot blot assay, we recommend to purify at least 20 μg of total RNA.  
    2. Determine the concentration of purified mRNA with NanoDrop and make a serial dilution of mRNA to 50 ng/μl, 10 ng/μl and 2 ng/μl using RNase-free water.
  2. Dot blotting
    1. Denature the serially diluted mRNA at 95 °C to disrupt secondary structures in a heat block for 3 min.
    2. Chill on ice immediately after denaturation to prevent the re-formation of secondary structures of mRNA.
    3. Drop 2 μl of mRNA directly onto the Hybond-N+ membrane optimized for nucleic acid transfer (Figure 1).

      Figure 1. Example of mRNA dots on a membrane

    4. Crosslink spotted mRNA to membrane in a Stratalinker 2400 UV Crosslinker twice using the Autocrosslink mode (1,200 microjoules [x100]; 25-50 sec).
    5. Wash the membrane in 10 ml of wash buffer in a clean washing tray, which is unnecessary to be RNase-free, for 5 min at room temperature with gentle shaking to wash off the unbound mRNA.
    6. Incubate the membrane in 10 ml of blocking buffer for 1 h at room temperature with gentle shaking.
    7. Incubate the membrane with anti-m6A antibody (1:250 dilution; 2 μg/ml) in 10 ml of antibody dilution buffer overnight at 4 °C with gentle shaking.
    8. Wash the membrane three times for 5 min each in 10 ml of wash buffer with gentle shaking.
    9. Incubate the membrane with goat anti-rabbit IgG-HRP (1:10,000 dilution; 20 ng/ml) in 10 ml of antibody dilution buffer for 1 h at room temperature with gentle shaking.
    10. Wash the membrane four times for 10 min each in 10 ml of wash buffer with gentle shaking.
    11. Incubate the membrane with 3 ml of ECL Western Blotting Substrate for 5 min in darkness at room temperature. Please note that the volume of ECL solution added is dependent on the size of the membrane. According to the manufacturer’s instructions, 0.125 ml ECL solution per cm2 of the membrane is recommended.
    12. Wrap the membrane in plastic wrap and expose with Hyperfilm ECL for a proper exposure period.
    13. Develop the film.

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.


This protocol is also applicable to detect other types of RNA modifications if the corresponding specific primary antibodies and secondary antibodies are available.


  1. Wash buffer
    1x PBS
    0.02% Tween-20
  2. Blocking buffer
    1x PBS
    0.02% Tween-20
    5% non-fat milk
  3. Antibody dilution buffer
    1x PBS
    0.02% Tween-20
    5% non-fat milk

Note: It is unnecessary to use RNase-free water to prepare the above solutions.


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.


  1. Fu, Y., Dominissini, D., Rechavi, G., and He, C. (2014). Gene expression regulation mediated through reversible m6A RNA methylation. Nat Rev Genet 15(5): 293-306.
  2. Shen, L., Liang, Z., Gu, X., Chen, Y., Teo, Z.W., Hou, X., Cai, W.M., Dedon, P.C., Liu, L., and Yu, H. (2016). N6-methyladenosine RNA modification regulates shoot stem cell fate in Arabidopsis. Dev Cell 38(2): 186-200.


N 6 - 甲基腺苷(m 6)是真核信使RNA(mRNA)的最普遍的内部修饰。可以通过几种方法检测m 6的总量,例如使用特异性m 5抗体的斑点印迹分析和定量液相色谱 - 串联质谱(LC- MS / MS)(Fu等人,2014; Shen等人,2016)。在这里,我们描述使用特异性m 5抗体通过斑点印迹分析来快速检测mRNA中的总m 6 A水平的方法。

背景 与其他方法(如二维薄层色谱和LC-MS / MS)相比,mRNA检测的斑点印迹分析相对容易,快速,经济有效。这种方法可以以定性的方式用于评估在不同发育阶段的各种植物组织或植物中的m

关键字:斑点印迹, RNA修饰, N6-甲基腺苷


  1. Amersham Hybond-N +膜(GE Healthcare,目录号:RPN203B)
  2. 保鲜膜
  3. > Amersham Hyperfilm ECL(GE Healthcare,目录号:28906835)
  4. 总RNA
  5. Dynabeads ® mRNA纯化试剂盒(Thermo Fisher Scientific,Ambion TM,目录号:61006)
  6. 无RNase的水
  7. 抗 - 抗体(Synaptic Systems,目录号:202 003)
  8. 山羊抗兔IgG-HRP(Santa Cruz Biotechnology,目录号:sc-2004)
  9. ECL Western Blotting Substrate(Thermo Fisher Scientific,Thermo Scientific TM ,目录号:32106)
  10. 1×磷酸缓冲盐水(1×PBS),pH7.4
  11. 吐温20(Sigma-Aldrich,目录号:P9416)
  12. 不含脂肪的牛奶(Bio-Rad Laboratories,目录号:1706404)
  13. 洗涤缓冲液(见配方)
  14. 阻塞缓冲区(见配方)
  15. 抗体稀释缓冲液(见配方)


  1. NanoDrop 2000分光光度计(Thermo Fisher Scientific,Thermo Scientific TM,型号:NanoDrop TM 2000/2000分光光度计)
  2. 热块
  3. Stratalinker 2400紫外线交联剂(Stratalinker)
  4. 振动器


  1. ImageJ


  1. mRNA纯化
    1. 使用Dynabeads mRNA纯化试剂盒按照制造商的说明书从总RNA中分离mRNA。对于一个斑点印迹测定,我们建议纯化至少20μg的总RNA。  
    2. 用NanoDrop确定纯化的mRNA的浓度,并使用无RNase的水进行mRNA的连续稀释至50 ng /μl,10 ng /μl和2 ng /μl。
  2. 斑点印迹
    1. 在95℃下使连续稀释的mRNA变性以破坏热块中的二级结构3分钟。
    2. 变性后立即在冰上冷却,以防止mRNA的二级结构重新形成。
    3. 将2μl的mRNA直接滴加到用于核酸转移优化的Hybond-N +膜上(图1)


    4. 交联通过自交联模式(1,200微焦[x100]; 25-50秒),在Stratalinker 2400紫外线交联剂中将膜上的膜分成两层。
    5. 将清洁的洗涤盘中的10毫升洗涤缓冲液中的膜在室温下清洗5分钟,清洗不需要RNase的洗涤盘,轻轻振荡洗涤未结合的mRNA。
    6. 在室温下温和摇动,将膜在10ml封闭缓冲液中孵育1小时。
    7. 将抗体(1:250稀释;2μg/ml)在抗体稀释缓冲液中于4℃温和摇动过夜孵育膜。
    8. 每次洗10分钟洗涤缓冲液,轻轻摇动膜3次,每次洗涤5分钟。
    9. 使用山羊抗兔IgG-HRP(1:10,000稀释; 20ng/ml)在10ml抗体稀释缓冲液中在室温下温和摇动孵育膜1小时。
    10. 每次洗10分钟10分钟洗涤缓冲液,轻轻摇动膜。
    11. 在室温下在黑暗中将3ml ECL Western Blotting底物孵育5分钟。请注意,添加的ECL溶液的体积取决于膜的尺寸。根据制造商的说明书,推荐使用0.125ml ECL溶液/cm 2。
    12. 将膜包裹在塑料包装中,并用超薄膜ECL曝光适当的曝光期。
    13. 制作电影


斑点印迹分析是半定量方法,应用独立生物材料通过上述步骤重复分析。与野生型对照相比,在独立材料中观察到的m 6 A水平的可重复变化被认为是"阳性"结果,可以通过其他定量方法进一步研究。此外,斑点印迹图像的信号可以通过ImageJ进行定量,统计分析应至少基于三个生物重复。


有关代表性的数据,请参见Shen 等人的论文,2016年。




  1. 洗涤缓冲液
    1x PBS
  2. 阻塞缓冲区
    1x PBS
  3. 抗体稀释缓冲液
    1x PBS





  1. Fu,Y.,Dominissini,D.,Rechavi,G.,and He,C.(2014)。  通过可逆的m6A RNA甲基化介导的基因表达调控。 Nat Rev Genet 15(5):293-306。
  2. Shen,L.,Liang,Z.,Gu,X.,Chen,Y.,Teo,ZW,Hou,X.,Cai,WM,Dedon,PC,Liu,L。和Yu,H。(2016) 。 N6-甲基腺苷RNA修饰调节芽干细胞命运拟南芥 Dev Cell 38(2):186-200。
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Copyright: © 2017 The Authors; exclusive licensee Bio-protocol LLC.
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.



Luigi Pasini
IRST - Scientific Institute of Romagna for the Study and Treatment of Cancer
Hi. I usually use total RNA as sort of positive control for a strong signal, in fact as said m6A is found in any RNA species. So, when I isolate mRNA (Dynabeads for polyA), the dot is less intense compared to the total RNA "control". I have extracted RNA with either Trizol or QUIAGEN/Norgen/Invitrogen kits and, for total RNA, I always had a good signal, typically with just 100-200 ng of RNA. In my experience, if you are looking for subtile changes in m6A, you must concentrate on the specific RNA species you are interested in, since of course just checking on total RNA might give no information.
8/3/2018 1:06:20 AM Reply
Ujunwa Okoye-Okafor
Just a question about the RNA purification procedure.

- Did anyone attempt this on total RNA and if yes how did you extract it?
Many thanks
8/2/2018 3:18:46 PM Reply
Yu Liu

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.

8/2/2018 3:32:39 PM

Hao Yu
Temasek Life Sciences Laboratory and Department of Biological Sciences, National University of Singapore, Singapore

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.

8/2/2018 6:52:12 PM

xueqin xu
5/18/2018 5:33:04 PM Reply