A New Method for Rapid Extraction of High Quality RNA from Recalcitrant Tissues of Grapevine

A quick, inexpensive, and reliable protocol for the extraction of RNA from grapevine berry skins containing large quantities of polyphenols, procyanidins, and polysaccharides is described. The method involves an extraction step in the presence of ribonuclease inhibitors and compounds that compete with vacuolar contaminants for binding to RNA. After extraction with organic solvents, RNA is bound to a fibrous cellulose matrix and processed to eliminate the remaining contaminants and ribonucleases. Following this method, highly stable RNA, sufficiently pure for northern hybridizations and enzymatic processing, may be obtained from as little as 200 mg of starting amounts of fresh material and without multiple, time consuming precipitations or ultracentrifugation steps. This procedure may also prove useful for extracting RNA from recalcitrant tissues of other plant species. Abbreviations: ATA, aurintricarboxylic acid; CF11, cellulose fibrous medium (type 11); PVPP, polyvinylpolypyrrolidone; RT room temperature; VRC, vanadyl ribonucleoside complex.     

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。     

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。     

一种从动物组织中提取高质量总RNA的方法

RNA提取技术是分子生物学研究中经常应用的最重要的实验技术。简要介绍了一种高纯度、高产量的从动物组织中提取总RNA的方法．该方法具有实用性强、重复性好的特点。提取的RNA无DNA等污染物,并且其产量、纯度完全能满足分子克隆和基因表达研究的需要。利用此方法提取牛组织的总RNA,进行了NRDR基因在牛组织中的表达分布研究。     

一种快速的胚胎组织总RNA的提取方法

简要介绍一种改进的提取人体器官组织总RNA的方法,该方法具有费用低,快速简便,重复性好的优点,提取的RNA无DNA等污染物,完全能满足基因表达研究的需要。     

一种高效经济的高质量植物RNA提取方法

建立了一种高效经济的植物RNA提取方法.在提取缓冲液中加入蔗糖、氯化钾和镁离子以提供对RNA分子的保护.破碎后的细胞于提取缓冲液中裂解后,用酚/氯仿变性并去除内源RNA酶和其他蛋白质,而后用pH 5.6 的NaAc沉淀RNA.用该方法提取RNA的得率较高,经电泳检测,RNA的完整性很好.RNA印迹分析和RT-PCR也都得到很好的结果.该方法还使实验成本大大降低.     

用低浓度硫氰酸胍提取高质量植物RNA

目前分离RNA的方法很多, 但大多是基于动物材料建立的方法, 而针对植物材料的方法并不多见. 建立了一种从植物材料中分离高质量RNA的硫氰酸胍/氯化锂/热酚法. 与其他硫氰酸胍的方法相比, 该方法所用硫氰酸胍的浓度仅为现有方法的1/40 (0.1 mol/L),降低了实验成本, 且所得RNA质量令人满意. 用该方法分离的RNA在琼脂糖凝胶电泳上可清晰分出4条核糖体RNA (rRNA)带; 用此RNA进行RNA印迹或分离mRNA进行体外翻译试验, 均获得很好效果.     

适用于微卫星标记的湿地松、加勒比松DNA快速提取法

以湿地松(Pinuselliottii,PEE),包括古巴加勒比松(P.caribaea var.caribaea,PCC)、洪都拉斯加勒比松(P.caribaea var.hondurensis,PCH)、巴哈马加勒比松(P.caribaeavar.bahmaensis,PCB)3个变种在内的加勒比松,侧枝顶芽为试验材料,使用RETSCHMM400混合球磨仪破碎植物组织,然后利用改良CTAB法提取基因组DNA,完成48个样品仅用1h,1个工作日可提取300个样品以上,DNA分子量大于20kb,0.3g样品可提取DNA20-60μg,能够满足几百次PCR反应;利用所提DNA进行SSR分析,条带清晰,多态性好,说明该方法提取的DNA完全可以满足SSR反应的需要。本研究为SSR标记用于湿地松、加勒比松分子标记辅助选择育种提供了经济、高效、可靠的DNA提取方法。     

猕猴桃RNA提取与RT-PCR

为了从富含多糖和多酚等物质的猕猴桃幼叶中提取和分离出高质量的RNA,用多个不同品种的猕猴桃叶为材料,比较了3种不同的RNA提取方法所提取的总RNA。结果表明,用胍-酚酸-DEPC法提取的RNA质量最好,提取率达到682.9~780.8μg?g(FW),其R值(A260?A280)接近1.90。用所提取的RNA样品进行RT-PCR,其扩增产物在琼脂糖凝胶上出现明显清晰的扩增cDNA带,说明RNA样品在纯度和浓度上都可以满足PCR等分子生物学实验的基本要求。     

高质量提取大豆叶片总RNA的改良方法

豆科植物总RNA的提取相对比较困难,大豆叶片富含多糖、蛋白质,传统方法提取效果均不理想.在总RNA的提取方法Trizol法和热酚-氯化锂沉淀法的基础上,针对大豆叶片的特殊性,做了适当的改良,从而获得了完整性好,高纯度的大豆叶片总RNA,并成功进行了基因片段的RT-PCR实验,结果证实所获得的RNA完全可以适应后续实验要求.     

富含多糖和次生物质的芒果子叶总RNA的提取

为了提取芒果子叶总RNA,去除次生物质和多糖等杂质的严重干扰,采用了正交试验快速优化提取方法。实验证明70%的丙酮能有效地去除大部分杂质,提取过程一天内可完成,RNA含量可达677μgg·FW,A260A280比值为195,RNA的纯度及质量适合后续反应。     

RNA extraction from plant tissues

Several protocols and commercial kits are used for the extraction of nucleic acids from different plant tissues. Although there are several procedures available to remove sugars, which hinder the extraction of clean genomic DNA, there are few to assist with extraction of RNA. Those presently used include precipitations with ethylene glycol monobutyl ether or lithium chloride (LiCl), or centrifugation in cesium chloride (CsCl) gradients, but these generally either do not allow high recovery of RNA, are time consuming, rely on hazardous chemicals or need special equipment. Here we present the use of the simple cation, Ca²⁺, which has been tested and shown to be very efficient for the precipitation of high molecular weight pectic sugars during RNA extraction. Results are presented for different plant tissues, especially tissues of peach and apple fruits at varying ripening stages.     

A method for isolating total RNA from pear leaves

Isolation of high quality RNA fromRosaceae species is particularly difficult. These plants contain considerable amounts of plant polyphenolic compounds and polysaccharides that copurify with RNA, often rendering it unsuitable for either cDNA synthesis and/or hybridization in northern analyses. We describe a method for RNA isolation from pear leaves that is modified from that of Manning (1990). The procedure includes i) an extraction with phenol and PVPP, to remove proteins and polyphenols ii) two purifications by LiCl, with a 2-butoxyethanol treatment between the LiCl steps. The method results in high quality RNA suitable for RT-PCR and northern blot experiments.     

从血液中提取总RNA的一种快速高效方法

血液中含有大量的RNA酶 ,可引起RNA的降解 .防止RNA酶的降解 ,是保证所得RNA片段完整的关键 .目前提取RNA的方法较多 ,但有些方法尚不能完全防止RNA降解 .将TRIZOL方法稍加改进 ,将TRIZOL与异硫氰酸胍联用提取血液淋巴细胞总RNA .琼脂糖凝胶电泳结果表明 ,其 2 8SRNA与 18SRNA的比值为 2∶1,优于单独使用其中任何一种试剂者 .此方法同样适用于从其它细胞中提取RNA .     

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。     

台湾牛樟总RNA提取方法的建立

以牛樟Cinnamomum kanehirae根、茎、叶为材料,在RNAprep Pure Plant Kit (Polysaccharides & Polyphenolics-rich)方法的基础上进行改良,建立台湾牛樟总RNA的提取方法。经琼脂糖凝胶电泳、Nanodrop和Aglient 2100检测发现,牛樟根、茎、叶所得RNA的28S和18S比值介于1.7～2.1之间,RNA完整性较好,浓度均在50 ng·μL-1以上,RIN值均大于7。该方法提取的RNA纯度、浓度和完整性均合格,可满足后续分子生物学实验要求。     

土壤水分交替变化对湿地松幼苗光合特性的影响

以盆栽湿地松幼苗为材料,通过实验室模拟三峡库区从水淹到干旱的过程,设置常规供水(CK)、常规供水-轻度干旱-复水(DR),水淹(FL),水淹-轻度干旱-复水(FD)4个实验组,研究土壤水分交替变化对湿地松幼苗光合参数及叶绿素含量的影响.结果表明:(1)不同土壤水分处理条件对湿地松幼苗的净光合速率、气孔导度、蒸腾速率、胞间CO2浓度、气孔限制值、水分利用效率和光合色素含量都有一定影响.(2)在实验的前中期,DR、FL和FD组湿地松幼苗的光合速率、气孔导度、蒸腾速率都显著下降,DR和FD在实验后期回升到较高水平.(3)在整个实验期间各处理组的水分利用效率较对照都有不同程度上升,并以DR组的最大(4.95μmol·mmol-1).(4)随着处理时间延长,各处理组湿地松幼苗的总叶绿素含量及叶绿素a/b值均呈先降后升的趋势,其中叶绿素/类胡萝卜素的比值在4.379～6.019间波动,而叶绿素a/b值则在2.207～2.850间波动.研究发现,湿地松幼苗在水分代谢、光合生理等方面等能够很好地适应水淹以及"淹-干"的水分交替变化环境,并且在解除胁迫后能够迅速恢复生长,可以考虑用于三峡库区消落带的植被修复和重建.     

An improved method for high-quality RNA isolation from needles of adult maritime pine trees

When conventional RNA isolation methods optimized for pine seedlings are applied to needles of adult pine trees, poor-quality RNA results. Here we describe a modified procedure to isolate high-quality RNA from needles of 30-year-old maritime pines, exhibiting high levels of phenolics, polysaccharides, and RNases. Major changes are the inclusion of proteinase K in the extraction medium followed by incubation at 42°C. Integrity and purity were evaluated by using denaturing gel electrophoresis and spectrophotometry (A₂₆₀/A₂₃₀ and A₂₆₀/A₂₈₀). The total RNA could be successfully used for poly(A)⁺-RNA isolation and cDNA library construction.     

A simple and effective method for isolating RNA from alfalfa pollen

Extraction of RNA from alfalfa pollen using conventional grinding devices resulted in low yields of degraded RNA; degradation appeared to be related to the length of time required to break open the pollen grains with such devices. A glass syringe was modified to provide the restricted, shearing environment necessary for rapidly rupturing this type of tissue. This apparatus would be useful for extracting a variety of molecules from pollen of any species, but is particularly valuable for species which produce low amounts of relatively small pollen grains.