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。     

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。     

一种快速提取小麦叶片总RNA的方法

从植物组织中提取高质量的RNA是进行植物分子生物学研究的必要前提和关键.同种植物不同器官的组织由于组成分的差异,提取RNA的方法也存在不同的难点.在苯酚法和氯化锂沉淀法的基础上,改进并提出了一种适合小麦叶片总RNA的快速提取方法,消除了蛋白质、DNA、多糖、多酚等污染.该方法提取的小麦叶片总RNA,完整性好、纯度高,可用于RT-PCR、N orthern杂交、RACE等实验操作,而且简单经济、快速、实验结果稳定,重复性好,还适合富含多糖和脂质的植物组织总RNA的提取.     

仿刺参体壁总RNA提取方法的建立

目的:通过对TRIzol一步法进行改进,建立一种从富含胶原蛋白、多糖及色素的仿刺参体壁提取总RNA的有效方法。方法:样品在液氮中研磨并用TRIzol匀浆后再进行抽提;对TRIzol一步法提取的总RNA进行DNaseⅠ消化和酚氯仿抽提,用2.5mol/L的醋酸钾沉淀,并加入适量糖原(10mg/mL)与RNA共沉淀。结果:琼脂糖凝胶电泳和紫外分光光度法以及RT-PCR检测结果表明,改进的方法能够有效去除基因组DNA、蛋白、多糖及色素的污染,RNA的产率提高。结论:制备的总RNA纯度高,完整性好,能够满足mRNA差异显示RT-PCR等分子生物学研究的要求,是一种提取仿刺参体壁及其他富含黏多糖、胶原蛋白和色素的动物组织总RNA的有效方法。     

一种适合从柑橘果皮提取总RNA的方法

柑橘果皮由于富含果胶、酚类物质等干扰RNA分离的物质,较难提取到纯度高的RNA。本试验建立了一种适于从柑橘果皮提取RNA的方法,从脐橙和蕉柑两种柑橘的果皮提取总RNA,经凝胶电泳、紫外分光光度法检测所提RNA的品质。研究结果表明,该法所提RNA条带清晰、无降解。OD260/OD280接近2.0,具有较高的纯度。RT-PCR试验结果进一步表明,该法提取的RNA纯度高,完全能够用于后续的分子生物学研究。     

一种简单高效的食用真菌总RNA提取方法

以金针菇为材料,建立了一种适合于富含RNase、多酚、多糖和糖蛋白的食用真菌RNA的提取方法,此方法在高浓度变性剂存在的条件下2次用苯酚-氯仿-异戊醇进行抽提去除DNA、蛋白质,并用异丙醇和乙酸钠选择性沉淀RNA、去除多糖,得到完整、均一的RNA样品。 Abstract:With Flammulina velutipes material,an improved method was developed for extracting total RNA from domestic fungus that are rich in RNase,polyphenols,polymeric carbohydrates and proteoglycans..Phenol-chloroform-isoamyl alcohol were used twice to clear DNA and protein under higher concentration of denaturing solution and isopentanol,sodium acetate were used to precipitate RNA selectively.Pure and intact RNA can be effectively prepared by this method.     

四种副溶血弧菌总RNA提取方法的比较

副溶血弧菌(Vibrio parahaemolyticus)是一种革兰氏阴性嗜盐菌.提取高质量的RNA是研究副溶血弧毒力基因表达与其致病性和环境适应性的基础.本研究分别用SDS法、Trizol法,PureLinkTM RNA Mini Kit和Uniq-10柱式Trizol总RNA抽提试剂盒提取六株副溶血弧菌的总RNA,用普通琼脂糖凝胶电泳和核酸测定仪检测RNA的质量,并用RT-PCR法对四种提取方法进行比较.研究结果表明,Trizol法和PureLinkTM RNA Mini Kit简单快捷,提取的总RNA完整性好,纯度高,可用于后续实验的分子生物学研究;而Trizol法更适用于普通实验室细菌RNA的提取.     

大型纤毛虫总RNA的提取方法

以冠突伪尾柱虫为材料,建立了一种适合于富含RNase和多糖的大型纤毛虫总RNA的提取方法。该方法采用异硫氰酸弧和β-巯基乙醇联合快速变性,酚、氯仿和异戊醇抽提,同时在变性剂存在的条件下两次选择性地沉淀RNA,从而有效地去除了DNA、蛋白质和多糖。所得RNA样品经电泳、紫外分光光度法检测和RT-PCR分析,证实为完整、均一且无基因组污染的总RNA.这为构建冠突伪尾柱虫有小核系与无小核系之间的削减文库、筛选两细胞系差异表达的基因奠定了基础。     

从茶树种胚中提取总RNA方法的研究

茶树种胚中富含多糖和多酚类化合物,这增加了总RNA的提取难度.试验以茶树种胚为研究对象,通过对几种提取总RNA方法的比较与分析,认为用改进的CTAB-LjCl法提取茶树种胚总RNA的产率较高,质量好,可直接用于cDNA的合成、cDNA-AFLP分析等分子生物学实验操作.     

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。     

Isolation of RNA from polysaccharide-rich seeds

Commonly, RNA isolation is the initial step in the study of gene expression analysis and also in the utilization of genes for genetic improvement. However, the recovery of large amounts of RNA with high quality is a difficult process, especially in tissues containing enhanced levels of polysaccharides and other secondary metabolites. Since several procedures for RNA isolation from polysaccharides rich plant tissues have been resulting in poor yields, an effective new protocol is essential for RNA isolation and further analysis. Here, we describe a novel modified technique for isolating total RNA from maturing grains. As a model, we utilized little finger millets, important food staples, which correspond to short duration crops cultivated in varied agro climatic conditions. After isolation, the total RNA was resolved on a denaturing agarose gel, showing more sharp bands of 28S, 18S, and 5S with no degradation. Therefore, the RNA concentration (higher than 1.80) was calculated by spectrophotometry, indicating that RNA is concentrated. Finally, RT-PCR and Northern hybridization confirmed high RNA quality.     

A method for RNA isolation from marine macro-algae

Sulfated, carboxylic polysaccharides and polyphenols found in marine macro-algae interfere with RNA isolation from these plants and inhibit RNA activities in vitro. Methods based on differential precipitation of RNA or carbohydrates in high salts were used to eliminate the acidic carbohydrates. To protect RNA from inactivation by oxidized polyphenols, strong reducing reagents were used to prevent polyphenol oxidation. RNA was successfully isolated from Macro-cystis pyrifera (brown alga), Porphyra schizophylla (red alga), and Enteromorpha intestinalis (green alga). mRNA isolated from the total RNA was shown to be translationally active.     

A Modified Protocol for RNA Extraction from Different Peach Tissues Suitable for Gene Isolation and Real-Time PCR Analysis

RNA extraction is the first step in the study of gene isolation and expression. However, it is difficult to extract high quantity and quality RNA from tissues containing large quantities of polysaccharides and polyphenols. Peach (Prunus persica), in addition to containing high levels of polysaccharides and polyphenols, is a challenging starting material for RNA isolation using a single method because of different amounts of those substances in diverse tissues. Based on three reported methods, we developed a modified RNA isolation protocol to solve this problem, leading to high quality and quantity of total RNA from peach mesocarp tissues of fruits which were sampled from all developmental stages and different storage periods, as well as from other tissues including flowers, leaves, stems, and roots. With our modified method, 28–650 μg of total RNA was routinely obtained from per gram of fresh material, gave at least a 1.16-fold improvement by compared with those isolated by other seven methods. The RNA extracts were successfully used in downstream applications such as RT-PCR, RACE, and real-time PCR.     

RNA Isolation method for polysaccharide rich algae: agar producing <Emphasis Type="Italic">Gracilaria tenuistipitata</Emphasis> (Rhodophyta)

RNA isolation is essential to study gene expression at the molecular level. However, RNA isolation is difficult in organisms (plants and algae) that contain large amounts of polysaccharides, which co-precipitate with RNA. Currently, there is no commercial kit available, specifically for the isolation of high-quality RNA from these organisms. Furthermore, because of the large amounts of polysaccharides, the common protocols for RNA isolation usually result in poor yields when applied to algae. Here we describe a simple method for RNA isolation from the marine red macroalga Gracilaria tenuistipitata var. liui Zhang et Xia (Rhodophyta), which can be applied to other plants and algae.     

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。     

RNA isolation from loquat and other recalcitrant woody plants with high quality and yield

RNA isolation is difficult in plants that contain large amounts of polysaccharides and polyphenol compounds. To date, no commercial kit has been developed for the isolation of high-quality RNA from tissues with these characteristics, especially for fruit. The common protocols for RNA isolation are tedious and usually result in poor yields when applied to recalcitrant plant tissues. Here an efficient RNA isolation protocol based on cetyltrimethylammonium bromide (CTAB) and two successive precipitations with 10 M lithium chloride (LiCl) was developed specifically for loquat fruits, but it was proved to work efficiently in other tissues of loquat and woody plants. The RNA isolated by this improved protocol was not only of high purity and integrity (A₂₆₀/A₂₈₀ ratios ranged from 1.90 to 2.04 and A₂₆₀/A₂₃₀ ratios were > 2.0) but also of high yield (up to 720 μg on average [coefficient of variation = 21%] total RNA per gram fresh tissue). The protocol was tested on loquat fruit (different stages of development, postharvest, ripening, and bruising), leaf, root, flower, stem, and bud; quince fruit and root; grapevine cells in liquid culture; and rose petals. The RNA obtained with this method is amenable to enzymatic treatments and can be efficiently applied for research on gene characterization, expression, and function.     

A Modified Protocol for RNA Isolation from High Polysaccharide Containing Cupressus arizonica Pollen. Applications for RT–PCR and Phage Display Library Construction

RNA isolation is the first step in the study of gene expression and recombinant protein production. However, the isolation of high quantity and high-quality RNA from tissues containing large amounts of polysaccharides has proven to be a difficult process. Cupressus arizonica pollen, in addition to containing high polysaccharide levels, is a challenging starting material for RNA isolation due to the roughness of the pollen grain’s walls. Here, we describe an improved technique for RNA isolation from C. arizonica pollen grains. The protocol includes a special disruption and homogenization process as well as a two-step modified RNA isolation technique which consists of an acid phenol extraction followed by a final cleanup using a commercial kit. Resulting RNA proved to be free of contaminants as determined by UV spectrophotometry. The quality of the RNA was analyzed on a bioanalyzer and showed visible 25S and 18S bands. This RNA was successfully used in downstream applications such as RT–PCR and phage display library construction.