Protocol: a beginner's guide to the analysis of RNA-directed DNA methylation in plants.
Protocol: a beginner's guide to the analysis of RNA-directed DNA methylation in plants.
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实验方案：植物中 RNA 指导的 DNA 甲基化分析的初学者指南。
DOI：
10.1186/1746-4811-10-18
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发表时间：
2014
期刊：
影响因子：
5.1
通讯作者：
Zhu JK
中科院分区：
文献类型：
--
作者：
Zhang H;Tang K;Wang B;Duan CG;Lang Z;Zhu JK
DNA 甲基化是一种保守的表观遗传标记，控制着许多真核生物的基因组稳定性、发育和环境反应。 DNA 甲基化可以由非编码 RNA 引导，包括小干扰 RNA 和支架 RNA。尽管 DNA 甲基化和调控非编码 RNA 的测量对于许多有兴趣探索其领域表观遗传调控的生物学家来说是理想的选择，但传统方法具有局限性并且在技术上具有挑战性。例如，传统的通过RNA杂交检测siRNA需要相对大量的小RNA，并且涉及放射性同位素。另一种方法是 RT-qPCR，在逆转录过程中使用茎环引物。然而，它需要一个先决条件，即应该知道 siRNA 的确切序列。通过使用模式生物拟南芥，我们开发了一种易于遵循的综合程序，用于高效、定量测量 DNA 甲基化、小干扰 RNA 和支架 RNA。从简化的核酸操作开始，我们使用 Chop PCR（甲基化敏感酶消化，然后进行 PCR）检查 DNA 甲基化水平，这可以快速筛选 DNA 甲基化突变体，而无需转基因报告基因。我们部署了一种简单的生物信息学方法来挖掘已发表的小RNA数据库，以获得感兴趣区域内单个24nt siRNA的核苷酸（nt）序列。随后对商业 TaqMan Small RNA Assay 的方案进行了优化，以实现对单个 siRNA 的可靠定量检测。我们使用巢式 qPCR 来量化低丰度且不含 Poly-A 尾部的支架 RNA。此外，细胞核部分能够将染色质相关支架 RNA 与染色质释放的同源非支架转录物分离。我们开发了一种对作为 RNA 指导的 DNA 甲基化核心成分的核酸进行定量研究的程序。我们的结果不仅证明了该方法的有效性，而且还提供了甲基化敏感限制性酶、新型 DNA 甲基化标记位点和相关 siRNA 序列的列表，所有这些对于未来的表观遗传学研究都很有价值。重要的是，详细提供了分步方案，以便表观遗传学经验很少的生物学家可以轻松遵循这些方法。
DNA methylation is a conserved epigenetic mark that controls genome stability, development and environmental responses in many eukaryotes. DNA methylation can be guided by non-coding RNAs that include small interfering RNAs and scaffold RNAs. Although measurement of DNA methylation and regulatory non-coding RNAs is desirable for many biologists who are interested in exploring epigenetic regulation in their areas, conventional methods have limitations and are technically challenging. For instance, traditional siRNA detection through RNA hybridization requires relatively large amount of small RNAs and involves radioactive isotopes. An alternative approach is RT-qPCR that employs stem loop primers during reverse transcription; however, it requires a prerequisite that the exact sequences of siRNAs should be known. By using the model organism Arabidopsis thaliana, we developed an easy-to-follow, integrative procedure for time-efficient, quantitative measurement of DNA methylation, small interfering RNAs, and scaffold RNAs. Starting with simplified nucleic acid manipulation, we examined DNA methylation levels by using Chop PCR (methylation-sensitive enzyme digestion followed by PCR), which allowed for fast screening for DNA methylation mutants without the need of transgenic reporters. We deployed a simple bioinformatics method for mining published small RNA databases, in order to obtain the nucleotide (nt) sequences of individual 24nt siRNAs within the regions of interest. The protocol of commercial TaqMan Small RNA Assay was subsequently optimized for reliable quantitative detection of individual siRNAs. We used nested qPCR to quantify scaffold RNAs that are of low abundance and without Poly-A tails. In addition, nuclei fraction enables separation of chromatin-associated scaffold RNAs from their cognate non-scaffold transcripts that have been released from chromatin. We have developed a procedure for quantitative investigations on nucleic acids that are core components of RNA-directed DNA methylation. Our results not only demonstrated the efficacy of this procedure, but also provide lists of methylation-sensitive restriction enzymes, novel DNA methylation marker loci, and related siRNA sequences, all of which can be valuable for future epigenetic studies. Importantly, step-by-step protocols are provided in details such that the approaches can be easily followed by biologists with little experience in epigenetics.
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影响因子：
48
作者：
Kasinathan, Sivakanthan;Orsi, Guillermo A.;Zentner, Gabriel E.;Ahmad, Kami;Henikoff, Steven
通讯作者：
Henikoff, Steven
影响因子：
14.8
作者：
Saleh, Abdelaty;Alvarez-Venegas, Raul;Avramova, Zoya
通讯作者：
Avramova, Zoya
影响因子：
14.9
作者：
Chen C;Ridzon DA;Broomer AJ;Zhou Z;Lee DH;Nguyen JT;Barbisin M;Xu NL;Mahuvakar VR;Andersen MR;Lao KQ;Livak KJ;Guegler KJ
通讯作者：
Guegler KJ
影响因子：
9.5
作者：
Zhang H;Zhu JK
通讯作者：
Zhu JK
DOI：
10.1101/sqb.2012.77.014936
发表时间：
2012
期刊：
Cold Spring Harbor symposia on quantitative biology
影响因子：
--
作者：
Zhang H;Zhu JK
通讯作者：
Zhu JK