一种高质量麦冬总RNA的提取方法

本方法采用CTAB作为去污剂,分别用氯仿/异戊醇反复抽提、LiCl沉淀,以去除蛋白质、碳水化合物和次生代谢物等杂质,用DNase处理去除DNA污染,最后用无水乙醇沉淀获得总RNA。该方法不仅能获得完整性好、纯度高的总RNA,而且操作简单、成本低廉、RNA产率高,对富含次生物质的中草药材植物组织总RNA的提取具有借鉴意义。     

大青杨叶片总RNA的快速提取方法

目的:寻求快速提取大青杨叶片总RNA的方法。方法:分别用改良CTAB法、改良SDS法、改良TRIzol法及某公司总RNA提取试剂盒提取大青杨叶片总RNA,并用紫外光谱分析、凝胶电泳方法对提取的总RNA进行鉴定。结果:用改良CTAB法和改良TRIzol法能有效地去除蛋白及多糖,提取到的总RNA纯度高,D260nm/D280nm分别为2.05和1.78,RNA的完整程度优于试剂盒法。结论:改良CTAB法为大青杨叶片总RNA的最佳提取方法。     

东亚砂藓(Rhacomitrum canescens)RNA提取方法的比较和改进

方法:以东亚砂藓为材料,用CTAB法、热硼酸盐法和改良的SDS/酸酚法提取东亚砂藓总RNA,并采用3种方法进行沉淀,从提取RNA的纯度、完整性、产量、耗时和RT-PCR效果等分析确定适用于东亚砂藓总RNA提取的方法。结果:研究表明:CTAB法提取的RNA 28S rRNA明显缺失,泳道模糊不清,杂质多,热硼酸法和改良的SDS/酸酚法提取RNA 28S rRNA,18S rRNA条带清晰,OD260/OD280都在1.7以上,纯度高,但热硼酸法提取出的RNA有DNA污染,RNA的含量(15.68~35.2μg.g-1)低于SDS/酸酚法提取RNA的含量(46.5~51.9μg.g-1)3种沉淀方法比较认为无水乙醇配合LiCl沉淀RNA节省时间,反转录和RT-PCR效果好。结论:改良的SDS/酸酚法适合东亚砂藓RNA的提取。     

自然干燥紫萼藓总RNA提取方法

介绍一种适合富含酚类、萜类等次生物质的干燥紫萼藓的总RNA的提取方法—SDS/酸酚法。采用SDS做为去污剂,用水饱和酚、氯仿和异戊醇进行抽提以去除蛋白、酚类等次生物质,醋酸钾和无水乙醇去除多糖等物质,最后LiCl沉淀获得总RNA。该方法不但获得了完整性好和纯度高的RNA,而且操作简单,成本也较低,对其他富含酚类、萜类等次生物质的干燥植物组织的总RNA的提取具有借鉴意义。     

巨桉叶片总RNA提取方法比较

本研究以巨桉嫩叶为材料,通过试剂盒法、改良CTAB法和改良SDS法,进行巨桉嫩叶总RNA提取方法的比较试验,探讨适合巨桉嫩叶总RNA提取的方法。通过超微量分光光度计、凝胶电泳和RT-PCR对总RNA提取质量进行检验,结果表明:改良CTAB和改良SDS法均能获得高质量的总RNA,条带清晰,纯度高,效果稳定,均适用于桉树叶片总RNA提取。     

小金海棠总RNA提取方法比较及cDNA的LD-PCR扩增

用CTAB和SDS-酚两种方法提取了小金海棠根的总RNA,以CTAB法提取的总RNA为模板,用长距离PCR(LD-PCR)的方法合成了双链cDNA并进行了扩增。结果表明,CTAB法比SDS-酚法提取的总RNA纯度高、完整性好,宜于进行RT-PCR和cDNA文库的构建,SDS-酚法操作简单易行,提取产物可以直接用于转膜进行Northern杂交。     

一种简易高效提取多种植物纯净DNA的方法

植物组织中次生代谢产物较多,要从中快速提取高质量的DNA比较困难。本文利用一种改良的CTAB法,通过在裂解后直接添加RNase A去除RNA污染,然后再经过一系列的抽提、沉淀、洗涤,获得纯净的DNA。经用琼脂糖凝胶电泳及核酸检测仪检测总DNA的纯度、浓度及质量。以提取的DNA为模版,PCR能够获得清晰的目的条带。结果表明通过该法可以简易、快速、高通量的提取多种植物的纯净DNA,为后续的分子生物学分析奠定了技术基础。     

茶树不同器官组织总RNA提取方法的研究

从茶树组织中提取高质量的总RNA,是开展茶树基因组学、功能基因组学研究的重要前提,而RNase、多酚类物质严重干扰茶树总RNA的分离提取。鉴于茶树组织总RNA提取过程难易不一、总RNA提取质量良莠不齐的现状,现对材料用量、提取液、DNA和蛋白质抽提液、RNA沉淀试剂、多酚氧化抑制剂等进行了比较研究,建立了一种适合茶树各器官组织总RNA提取的简单高效的方法(简易CTAB-LiCl法),并与实验室常用的改良Tri-Reagent法、改良CTAB法进行了比较。核酸定量和琼脂糖凝胶电泳检测结果显示,简易CTAB-LiCl法从茶树各器官组织中提取到的总RNA质量高、得率高。总RNA的得率是改良CTAB法的1.6-5倍。因此,简易CTAB-LiCl法具有效率高、适用范围广,且操作简单、实验成本低的特点。RT-PCR和cDNA-AFLP实验表明,提取的总RNA能够用于后续的分子生物学研究。     

盐生肉质化植物海马齿(Sesuvium portulacastrum L.)中总RNA提取方法的优化

以海马齿为材料,分别用CrAB法、SDS法、Trizol方法以及改进的CTAB法提取其总RNA,并比较了各RNA的产率、纯度和完整性等.结果表明,改进的CrAB法对海马齿总RNA的提取有较好的效果.所得总RNA的28S、18S和5S条带清晰,A260/A280比值为2.0,A260/A230比值为2.08,RNA产量可达56μg·g-1(FW).经RT-PCR获得了特异条带,说明利用改良的CTAB法从海马齿中提取到的RNA质量好、产率高、完整性强,完全适合于进一步的分子生物学研究.     

淡水育珠蚌外套膜提取总RNA的改良方法

通过对Trizol法加以改进,提取淡水珍珠蚌外套膜组织中的总RNA。经预处理后,在异丙醇沉淀RNA时加入高浓度的盐溶液,用75%的酒精2次洗涤RNA。用紫外分光光度法和1%琼脂糖凝胶电泳鉴定所提取的RNA。结果表明,改良法获得的总RNA完整、纯度高,改良Trizol法是一种从淡水育珠蚌外套膜组织中提取总RNA的高效、便捷、可靠的方法。     

一种从苏铁叶片中有效提取RNA的方法

由于苏铁（ Cycas revoluta） 叶片中含有大量的多糖多酚等次生代谢物, 常规RNA 提取方法很难获得优质的RNA。在常规的CTAB 法中加入了硼砂和β- 巯基乙醇来消除多酚和多糖的干扰, 得到了一个从苏铁叶片中有效提取RNA 的方法, 每克鲜叶片可获得约930μg RNA。A260 280 和A260 230 的纳米波长的吸收比值都约为2 , 表明RNA 的质量较好。获得的RNA 可用于Northern blot 和反转录PCR 等分析, 也说明RNA 的质量比较好。此外, 改进的提取方法也适合于含有次生代谢产物的其它植物, 同样可以获得优质RNA。     

Rapid isolation of high-quality total RNA from taxus and ginkgo

An easy and efficient protocol was developed for isolating good-quality total RNA from various tissues including fruits, leaves, stems, and roots of ancient gymnosperm species, taxus and ginkgo. The protocol was developed based on the CTAB method with modifications, including higher-strength CTAB to help the lysis of plant cells, more PVP, and beta-mercaptoethanol to prevent oxidation of phenolic complexes, and higher-centrifugation force to get rid of most cell debris and to ensure RNA quality. In RNA isolation, chloroform/isoamyl alcohol was used to remove proteins, genomic DNA, and secondary metabolites and lithium chloride was subsequently adopted to concentrate total RNA away from most of the cytoplasmic components. Good-quality total RNA from various tissues of native taxus and ginkgo could be easily isolated within 24 hr by this protocol which avoided the limitation of plant materials and the usage of dangerous chemicals, such as phenol, and could provide total RNA for all kinds of further molecular studies.     

三七总RNA提取方法的对比研究

比较利用改进的异硫氰酸胍一步法、异硫氰酸胍高盐法、CTAB法和Thomas’RNA提取法等4种方法提取三七根茎2个部位总RNA的可行性。结果表明,改进的异硫氰酸胍一步法和异硫氰酸胍高盐法能有效地抑制酚类物质、多糖及皂苷等次级代谢产物对总RNA的影响,可从三七根茎中获得质量高、完整性好的总RNA。RT—PCR分析显示提取的总RNA具有反转录活性。这2种方法具有快速、简单、有效的特点。     

A reliable and efficient method for total rna isolation from various members of spurge family (Euphorbiaceae)

Introduction – It is prerequisite and crucial to extract RNA with high quality and integrity in order to carry out molecular biology studies in any plant species of a family. Euphorbiaceae members are known for high levels of their waxes, oils with polysaccharides, polyphenolics and secondary metabolites. These conditions are recognised to interfere unfavourably with various methodologies of RNA isolation. Objective – To develop a simple, rapid and reproducible cetyltrimethylamonium bromide (CTAB)‐based protocol, to reduce the time and cost of extraction without reducing quality and yield of RNA extracted from various recalcitrant Euphorbiaceae member plant tissues such as from tree leaves (Hevea brasilensis), woody shrubs leaves (Ricinus communis, Jatropha curcas, Manihot esculenta) and storage root tissue (M. esculenta). Methodology – Simple modifications and fast steps were introduced to the original CTAB protocol. All centrifugation steps were carried out at 4°C at 12000 rpm for 10 min, the sample weight was decreased and usage of spermidine and LiCl was omitted, reducing incubation time prior to RNA precipitation. This rapid CTAB protocol was compared with various RNA isolation methods intended for use with plants rich in polysaccharides and secondary metabolites. Results – The procedure can be completed within 2 h and many samples can be processed at the same time. RNA of high quality could be isolated from all the tissues of species that we tried. The isolated RNA from different species served as a robust template for RT‐PCR analysis. Conclusion – The study has shown that the improvement of a CTAB‐based protocol allows the rapid isolation of high‐quality RNA from various recalcitrant Euphorbiaceae members. Copyright © 2010 John Wiley & Sons, Ltd.     

Development of an Efficient Protocol of RNA Isolation from Recalcitrant Tree Tissues

Isolation of RNA from recalcitrant tree tissues has been problematic due to large amounts of secondary metabolites and interfering compounds in their cells. We have developed an efficient RNA extraction method, which yielded high-quality RNA preparations from tissues of the lychee tree. The method reported here utilized EDTA, LSS, and CTAB to successfully inhibit RNase activities. It was found that a high ionic strength brought about by 2 M NaCl was necessary. In addition, secondary metabolites and other interfering compounds were effectively removed using sodium borate and PVPP under a deoxidized condition. The quality of purified RNA was tested by both RACE and Northern blotting analysis, ensuring that the RNA could be used for subsequent gene expression analysis. This method has been successfully applied to purify RNA from 15 other plant species. In conclusion, the protocol reported here is expected to have excellent applications for RNA isolation from recalcitrant plant tissues.     

改良CTAB法提取林木树种基因组DNA的研究

目的:验证改良CTAB法提取不同林木树种基因组DNA的效果,寻求一种对于不同林木树种基因组DNA提取普遍使用的方法。方法:采用改良的CTAB法提取22种木本植物基因组DNA,并对其进行定性、定量分析及酶切分析。结果:采用本法可以去除多糖和其他次生代谢物并获得高质量的DNA。结论:该方法可以作为一种适于在实验室进行的林木树种基因组DNA的提取方法。