搜索

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   

下载 PDF 引用 收藏 提问与回复 分享您的反馈 Cited by

本文章节

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

  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)

Equipment

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

Software

  1. ImageJ

Procedure

  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.

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

  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.

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

  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.

简介

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
A水平的时间和空间变化。这对于通过其他复杂和定量方法进行详细调查之前初步检查相关突变体中m
A水平的变化特别有用。

关键字:斑点印迹, RNA修饰, N6-甲基腺苷

材料和试剂

  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)

设备

  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

软件

  1. ImageJ

程序

  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)

      图1.膜上mRNA点的实例

    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

数据分析

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。

代表数据

有关代表性的数据,请参见Shen 等人的论文,2016年。

笔记

如果相应的特异性一级抗体和二级抗体可用,该方案也适用于检测其他类型的RNA修饰。

食谱

  1. 洗涤缓冲液
    1x PBS
    0.02%吐温-20
  2. 阻塞缓冲区
    1x PBS
    0.02%Tween-20
    5%无脂牛奶
  3. 抗体稀释缓冲液
    1x PBS
    0.02%Tween-20
    5%无脂牛奶

注意:无需使用无RNase的水来准备上述解决方案。

致谢

这项工作得到了新加坡教育部新加坡国家研究基金会调查计划(NRF-NRFI2016-02)的学术研究基金(MOE2015-T2-1-002)和新加坡国立大学的校内研究支持和淡马锡生命科学实验室。

参考文献

  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。
  • English
  • 中文翻译
免责声明 × 为了向广大用户提供经翻译的内容,www.bio-protocol.org 采用人工翻译与计算机翻译结合的技术翻译了本文章。基于计算机的翻译质量再高,也不及 100% 的人工翻译的质量。为此,我们始终建议用户参考原始英文版本。 Bio-protocol., LLC对翻译版本的准确性不承担任何责任。
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.
提问与回复

(提问前,请先登录)bio-protocol作为媒介平台,会将您的问题转发给作者,并将作者的回复发送至您的邮箱(在bio-protocol注册时所用的邮箱)。为了作者与用户间沟通流畅(作者能准确理解您所遇到的问题并给与正确的建议),我们鼓励用户用图片或者视频的形式来说明遇到的问题。由于本平台用Youtube储存、播放视频,作者需要google 账户来上传视频。

当遇到任务问题时,强烈推荐您提交相关数据(如截屏或视频)。由于Bio-protocol使用Youtube存储、播放视频,如需上传视频,您可能需要一个谷歌账号。