Rapid and efficient isolation of high-quality small RNAs from recalcitrant plant species rich in polyphenols and polysaccharides.
Rapid and efficient isolation of high-quality small RNAs from recalcitrant plant species rich in polyphenols and polysaccharides.
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DOI：
10.1371/journal.pone.0095687
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发表时间：
2014
期刊：
影响因子：
3.7
通讯作者：
Fan Z
中科院分区：
文献类型：
--
作者：
Peng J;Xia Z;Chen L;Shi M;Pu J;Guo J;Fan Z
小rna包括microRNAs （miRNAs）和小干扰rna (sirna)，是植物发育和基因表达的重要调控因子。高质量小rna的获取是其表达和功能分析研究的第一步，但在具有各种次生代谢物的顽固植物组织中提取小rna的方法尚不完善，特别是对于富含多糖和多酚的热带和亚热带植物物种。在这里，我们开发了一种简单有效的方法，从顽固的植物物种中提取高质量的小rna。在RNA分离之前，CTAB-PVPP缓冲系统的预处理步骤可以有效地去除匀浆裂解物中干扰RNA的化合物和次级代谢物。然后，用Trizol试剂提取总rna，然后用聚乙二醇（PEG） 8000对高分子量（HMW） rna进行差异沉淀。最后，小rna可以很容易地通过乙醇沉淀从上清中回收，而不需要额外的消除步骤。与其他已发表的方案相比，从木瓜中分离的小rna通过清晰的凝胶背景和miR159a探针明显的北方印迹信号显示出高质量。此外，从木瓜中提取的小rna成功地用于验证预测的mirna和假定的保守的tasiARFs。此外，本文所描述的提取方法也在其他几种亚热带和热带植物组织中进行了试验。分离的小rna的纯度足以分别用于末端茎环RT-PCR和northern印迹分析等应用。本文报道的简单可行的提取方法有望在从富含多酚和多糖的顽固植物组织中分离小rna方面具有良好的潜力。
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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影响因子：
14.9
作者：
Kozomara A;Griffiths-Jones S
通讯作者：
Griffiths-Jones S
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14.9
作者：
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作者：
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影响因子：
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作者：
Cheng Han, Cheng Han;Gao Jing, Gao Jing;Huang, H. S.
通讯作者：
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影响因子：
5.1
作者：
Rosas-Cárdenas Fde F;Durán-Figueroa N;Vielle-Calzada JP;Cruz-Hernández A;Marsch-Martínez N;de Folter S
通讯作者：
de Folter S