CTAB法提取中草药材滇紫草细胞的总RNA

介绍一种适合富含次生产物的中草药材滇紫草细胞总RNA的提取方法——CTAB法。采用CTAB作为去污剂,分别用氨仿和水饱和酚反复抽提以及LiCl沉淀以去除蛋白质、碳水化合物和次生代谢物等杂质,最后用纯乙醇沉淀获得RNA。该方法不但能获得完整性好和纯度高的总RNA,而且操作简单、快速、成本低廉,对其它富含次生物质的植物特别是中草药材组织总RNA的提取具有借鉴意义。     

一种从富含多糖的玉米幼穗中提取RNA的方法

介绍了一种大量提取玉米(Zea mays L.)幼穗总RNA的有效方法。由于玉米幼穗富含多糖, 用普通的RNA提取方法和Trizol很难获得高质量的RNA。在热酚法提取RNA的基础上, 通过在提取缓冲液中加入低pH值的醋酸钾来去除多糖。所得到 的RNA获得率高, 质量好, 条带完整, 实验证明可直接用于RT-PCR、微阵列等各项后续分子生物学实验。     

一种从富含多糖的玉米幼穗中提取RNA的方法

介绍了一种大量提取玉米(Zeamays L.)幼穗总RNA的有效方法。由于玉米幼穗富含多糖,用普通的RNA提取方法和Trizol很难获得高质量的RNA。在热酚法提取RNA的基础上,通过在提取缓冲液中加入低pH值的醋酸钾来去除多糖。所得到的RNA获得率高,质量好,条带完整,实验证明可直接用于RT-PCR、微阵列等各项后续分子生物学实验。     

茶树叶片总RNA提取方法的比较研究

针对茶树叶片中富含多酚类物质的特点,以陕西地方茶树代表种质紫阳槠叶种的叶片为材料,比较了Trizol法、异硫氰酸胍法和改良SDS-酸酚法三种不同的总RNA提取方法。结果表明改良SDS-酸酚法能够从新鲜叶片、冷冻叶片和干燥叶片中提取到质量高、完整性好的总RNA,经检测28SrRNA亮度为18SrRNA的2倍,A260/A280值介于1.83～2.01之间。以鲜叶、冻叶和干燥叶片的总RNA为材料,进一步用于DDRT-PCR分析,均可获得清晰稳定的多态性结果。说明改良的SDS-酸酚法能抑制茶树叶片中多酚类物质对总RNA提取的影响,是一种适于茶树叶片总RNA提取的有效方法。     

干种子高质量总RNA的快速提取方法

高效快速提取高质量的种子RNA是种子分子生物学研究的基础。现有的提取方法难以高效快速地从种子中得到高质量的总RNA。本试验有机地将改进SDS法和异硫氰酸胍法相结合,采用改进的酸性SDS提取液、不溶性PVPP(聚乙烯聚吡咯烷酮)阻止酚类氧化、KAc去除多糖、异丙醇沉淀RNA,可以高效地从0.01～0.1g水稻、大豆、蚕豆、芸豆、花生等干种子中提取到高质量总RNA。此法提取的总RNA,能够满足分子生物学研究的要求,可以进行反转录和RT-PCR反应,用于基因表达研究,并为从具相似成分的其他物种干种子提取总RNA提供参考方法。     

一种高效提取棉花RNA的改良方法

与其他植物相比,棉花组织中含有较多的酚类、萜类和多糖等次生代谢物,这些物质严重影响棉花细胞中RNA的分离。针对棉花富含次生代谢物的特点,同时简化操作步骤,摸索出一种高效提取棉花RNA的方法,该方法具有提取RNA完整性好、纯度高和操作简单等特点,可适用于提取棉花等多糖多酚类植物的RNA。     

提取蕨类植物蜈蚣草总RNA 的一种有效方法

介绍了一种提取蕨类植物蜈蚣草(Pteris vittata L.)总RNA的有效方法。由于蜈蚣草富含多酚和多糖,用普通的RNA提取方法很难获得高质量的RNA。本方法通过优化提取缓冲液的条件来抑制多酚氧化,并利用RNA与多酚、多糖在不同浓度的2-丁氧乙醇中溶解度不同的特性来去除多酚和多糖。本方法简便、快速,所提取的RNA质量较高,可直接用于cDNA合成、cDNA文库的构建以及RACE等分子生物学操作。     

提取蕨类植物蜈蚣草总RNA的一种有效方法

介绍了一种提取蕨类植物蜈蚣草(Pteris vittata L.)总RNA的有效方法.由于蜈蚣草富含多酚和多糖,用普通的RNA提取方法很难获得高质量的RNA.本方法通过优化提取缓冲液的条件来抑制多酚氧化,并利用RNA与多酚、多糖在不同浓度的2-丁氧乙醇中溶解度不同的特性来去除多酚和多糖.本方法简便、快速,所提取的RNA质量较高,可直接用于cDNA合成、cDNA文库的构建以及RACE等分子生物学操作.     

萝卜总RNA提取与mRNA差异显示技术

以萝卜幼小花药与叶片为材料,初步建立了mRNA差异显示技术体系.4种提取总RNA方法中,改进的SDS/酸酚法比CTAB/酸酚法和SDS/碱酚法更合适;改进的热硼酸法(HB)提取的RNA质量也较高,但所需时间较长,成本较高.以改进的SDS/酸酚法获得的RNA进行纯化后,其OD260/OD280在2.0～2.2之间,表明RNA样品杂质较少;总RNA进行反转录与差异显示分析表明,高分辨力的cDNA片段在100～380 bp之间.     

莲藕组织总RNA的快速提取方法

莲藕组织富含多糖、脂质、酚类等物质,用一般的方法较难提取高质量的RNA。在改进前人方法的基础上,建立了一种高效、简单的CTAB-LiCl提取法,能快速提取高质量的莲藕组织总RNA,并且产率高、完整性好、纯度高,能进一步满足RT-PCR等分子生物学实验的需要。此外,该方法也适用于其它富含多糖、脂质、酚类等物质的植物组织总RNA的提取。     

何首乌总RNA提取方法的比较及改进

目的：探讨不同提取方法对提取何首乌总RNA的质量影响,寻找适于何首乌成熟叶组织RNA的提取方法。方法：以何首乌成熟叶组织为材料,采用SDS／酸酚法、常规CTAB法及改良的TRIzol试剂法分别进行实验,并对所提取RNA的质量进行验证。结果：采用3种方法都能提取出RNA,但质量差异较大。其中改良的TRIzol试剂法能有效抑制次生物质的影响,提取的RNA产量可达70-110μg／g,纯度高于其他2种方法,D260nm／D280nm值为1．85～1．97。结论：改良的TRIzol试剂法操作简便,提取的RNA完整性和纯度较高,可以满足下一步实验的要求。     

An improved method for the isolation of total RNA from Avicennia germinans leaves

Isolation of high-quality RNA of Avicennia germinans L. tissue is difficult due to high levels of phenols and other substances that interfere when using conventional procedures for the isolation. These substances not only decrease the yield but also the quality of RNA is almost poor. We present here a simple RNA protocol and fast methodology that effectively removes these contaminating substances without affecting the yield. The protocol developed is based on the SDS/phenol method with modifications including beta-mercaptoethanol to prevent oxidation of phenolic complexes, and phenol/chloroform extraction is introduced to remove proteins, genomic DNA, and secondary metabolites, and co-precipitated polysaccharides. Both A260/A230 and A260/A280 absorbance ratios of isolated RNA were around 2 and the yield was about 0.3 mg g(-1) fresh weight. Good-quality total RNA from leaves of Avicennia germinans could be easily isolated within 2 h by this protocol which avoided the limitation of plant materials and could provide total RNA for all kinds of further molecular studies.     

麦冬根中总RNA的快速提取

目的:从富含多糖、多酚的麦冬根部组织中快速提取总RNA。方法:采用改进的苯酚法,提取液的配制为5%SDS、1mol/LNaAc(pH4.1)、20%HAC、0.1%PVP。结果:采用该方法提取的麦冬总RNA纯度高、完整性好,电泳条带清晰。通过琼脂糖凝胶电泳与紫外吸光度测定产量与纯度,麦冬根部组织总RNA的吸光值D260nm/D280nm值大于1.8,D260nm/D230nm值大于2.0,麦冬块根与不定根RNA的平均产量分别为79.716和76.144μg/g(鲜重)。结论:用本方法提取的RNA可用于后继的抑制消减杂交试验。     

Extensive deproteinization of Dictyostelium discoideum RNase P reveals a new catalytic activity.

Nuclear Dictyostelium discoideum RNase P was subjected to vigorous deproteinization procedures. After treatment with proteinase K followed by phenol extraction of samples containing D. discoideum RNase P activity, a new enzymatic activity was recovered. The proteinase K/phenol/SDS treated enzyme cleaves Schizossacharomyces pombe tRNAser (supS1), D. discoideum tRNASer and tRNALeu precursors several nucleotides upstream of the cleavage site of RNase P, liberating products with 5'-hydroxyl ends. This activity seems to be associated with one or two RNA molecules copurifying with D. discoideum RNase P activity as judged by its inhibition in the presence of micrococcal nuclease, which is in contrast to its resistance to proteinase K/phenol/SDS treatment.     

Isolation and purification of functional total RNA from blue-grained wheat endosperm tissues containing high levels of starches and flavonoids

Isolation of intact, functional total RNA from blue-grained wheat endosperms containing high levels of starches, polysaccharides, and flavonoids was extremely difficult using traditional methods. We describe here a modified SDS/phenol method that can be used to isolate total RNA from blue-grained wheat endosperms. This method solved the problems of RNA degradation, contamination, and low yield due to binding and/or co-precipitation with starches, polysaccharides, and flavonoids. The isolated total RNA was of high quality and undegraded as determined by spectrophotometric readings and denaturing agarose gel analysis. Quality of the total RNA isolated using our protocol was further assessed by RT-PCR and northern blot hybridization. Using this efficient procedure, 350–400 μg of total RNA was routinely obtained from 1 g of blue-grained wheat seeds.     

Measurements of DNA and RNA in mammary gland homogenates by the ethidium bromide technique.

A rapid method of determining simultaneously DNA and RNA in mammary gland homogenates using the ethidium bromide technique is discussed. The method utilizes a quantitative extraction of DNA and RNA with 2.0m sodium chloride, SDS, and EDTA at pH 8.0. Assays of mammary gland RNA and DNA using previously published methods were compared with determinations using the ethidium bromide technique. While the fluorescence method gave lower values for RNA when compared to those obtained using the orcinol or absorbancy ratio (OD 260nm/280nm), DNA measurements agreed well with the values determined by the diphenylamine technique. Extinction coefficient data for total mammary gland RNA isolated using a modified phenol extraction procedure are also presented.     

快速提取棉花蕾期高质量RNA的改良方法

棉花组织中因富含棉酚、多糖和单宁等次生代谢物,RNA难分离,易降解,导致现有RNA快速提取试剂盒提取的棉花RNA产率低、质量差,无法应用于实验操作。针对棉花组织次生代谢物多的特点,以酸酚法为基础,结合RNA吸附柱,通过对幼苗期和蕾期棉花叶片RNA提取,总结出一种快速提取棉花组织RNA的方法。与热硼酸法、酸酚法及试剂盒法相比,所述方法具有步骤少、产率高、质量好等特点,操作简单、整个实验在1h内完成,可开发出提取多糖多酚类生物组织RNA试剂盒。