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Rapid and Efficient Isolation of High-Quality Small RNAs from Recalcitrant Plant Species Rich in Polyphenols and Polysaccharides

文献类型： 外文期刊

作者： Peng, Jun 1 ; Xia, Zihao 1 ; Chen, Ling 1 ; Shi, Minjing; Pu, Jinji 3 ; Guo, Jianrong 4 ; Fan, Zaifeng 1 ;

作者机构： 1.China Agr Univ, State Key Lab Agrobiotechnol, Beijing 100094, Peoples R China

2.China Agr Univ, Minist Agr Key Lab Plant Pathol, Beijing 100094, Peoples R China

3.Chinese Acad Trop Agr Sci, Environm & Plant Protect Inst, Minist Agr, Key Lab Integrated Pest Management Trop Crops, Haikou, Hainan, Peoples R China

4.Chinese Acad Trop Agr Sci, Environm & Plant Protect Inst, Minist Agr, Key Lab In

期刊名称：PLOS ONE （ 影响因子：3.24； 五年影响因子：3.788 ）

ISSN： 1932-6203

年卷期： 2014 年 9 卷 5 期

页码：

收录情况： SCI

摘要： Small RNAs, including microRNAs (miRNAs) and small interfering RNAs (siRNAs), are important regulators of plant development and gene expression. The acquisition of high-quality small RNAs is the first step in the study of its expression and function analysis, yet the extraction method of small RNAs in recalcitrant plant tissues with various secondary metabolites is not well established, especially for tropical and subtropical plant species rich in polysaccharides and polyphenols. Here, we developed a simple and efficient method for high quality small RNAs extraction from recalcitrant plant species. Prior to RNA isolation, a precursory step with a CTAB-PVPP buffer system could efficiently remove compounds and secondary metabolites interfering with RNAs from homogenized lysates. Then, total RNAs were extracted by Trizol reagents followed by a differential precipitation of high-molecular-weight (HMW) RNAs using polyethylene glycol (PEG) 8000. Finally, small RNAs could be easily recovered from supernatant by ethanol precipitation without extra elimination steps. The isolated small RNAs from papaya showed high quality through a clear background on gel and a distinct northern blotting signal with miR159a probe, compared with other published protocols. Additionally, the small RNAs extracted from papaya were successfully used for validation of both predicted miRNAs and the putative conserved tasiARFs. Furthermore, the extraction method described here was also tested with several other subtropical and tropical plant tissues. The purity of the isolated small RNAs was sufficient for such applications as end-point stem-loop RT-PCR and northern blotting analysis, respectively. The simple and feasible extraction method reported here is expected to have excellent potential for isolation of small RNAs from recalcitrant plant tissues rich in polyphenols and polysaccharides.

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