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Zhang H, Tang K, Wang B, et al. Protocol: a beginner's guide to the analysis of RNA-directed DNA methylation in plants. Plant Methods. 2014;10:18doi: 10.1186/1746-4811-10-18.
Zhang, H., Tang, K., Wang, B., Duan, C. G., Lang, Z., & Zhu, J. K. (2014). Protocol: a beginner's guide to the analysis of RNA-directed DNA methylation in plants. Plant methods, 1018. https://doi.org/10.1186/1746-4811-10-18
Zhang, Huiming, et al. "Protocol: a beginner's guide to the analysis of RNA-directed DNA methylation in plants." Plant methods vol. 10 (2014): 18. doi: https://doi.org/10.1186/1746-4811-10-18
Zhang H, Tang K, Wang B, Duan CG, Lang Z, Zhu JK. Protocol: a beginner's guide to the analysis of RNA-directed DNA methylation in plants. Plant Methods. 2014 Jun 14;10:18. doi: 10.1186/1746-4811-10-18. eCollection 2014. PMID: 24955108; PMCID: PMC4065543.
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Plant Methods. 2014 Jun 14;10:18. doi: 10.1186/1746-4811-10-18. eCollection 2014.

Protocol: a beginner's guide to the analysis of RNA-directed DNA methylation in plants.

Plant methods

Huiming Zhang, Kai Tang, Bangshing Wang, Cheng-Guo Duan, Zhaobo Lang, Jian-Kang Zhu

Affiliations

  1. Department of Horticulture and Landscape Architecture, Purdue University, West Lafayette, IN 47907, USA.
  2. Department of Horticulture and Landscape Architecture, Purdue University, West Lafayette, IN 47907, USA ; Shanghai Center for Plant Stress Biology, Shanghai Institute of Biological Sciences, Chinese Academy of Sciences, Beijing, Shijingshan, China.

PMID: 24955108 PMCID: PMC4065543 DOI: 10.1186/1746-4811-10-18

Abstract

BACKGROUND: 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.

RESULTS: 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.

CONCLUSIONS: 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.

Keywords: Epigenetics; Nuclei fractionation; Pol IV; Pol V; RNA-directed DNA methylation; Scaffold RNA; siRNA

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