Comparison of RNA Extraction Methods of Sugarcane Stem

[Objective] The study was to explore the most effective and feasible method for sugarcane stalks RNA extraction.[Method] SDS extraction method,kit extraction method and GHCL extraction method were used to extract the sugarcane RNA in stalks,and the quality of the extracted RNA was compared.[Result] RNA extracted by kit extraction method had a high-yield,the bands were clear and RNA had a good integrity,there was no significant degradation of RNA,and the OD260 nm/OD280 nm value was closed to 2.0.[Conclusion] Kit extraction method was the effectively method to extract sugarcane RNA,and this study had provided a theoretical basis for the molecular biology study of sugarcane.

Study on Extraction of Effective Aroma Components from Formula Tobacco by the Supercritical CO2 Extraction Method and Its Aroma Composition

[Objectives]This study was conducted to improve the sensory quality and industrial availability of tobacco extracts.[Methods]The L9(34)design was adopted to carried out an extraction experiment,in which formula tobacco was extracted using supercritical CO2,and the extract was concentrated by vacuum distillation.Through sensory evaluation and chemical analysis,the function determination and chemical composition analysis of the tobacco extracts were carried out,and the optimal supercritical fluid extraction process was finally determined.[Results]The obtained optimal supercritical fluid extraction conditions were as follows:extraction temperature 55℃,extraction pressure 25 MPa,CO2 flow rate of 20 L/h,and entrainer of 95%ethanol.The tobacco extract obtained under the optimal conditions endowed the cigarettes with full and delicate aroma,less irritation and clean aftertaste and made the flavor of the cigarettes overall coordinated and softer,so the sensory quality was significantly improved.[Conclusions]The tobacco extract obtained by the supercritical CO2 extraction method from formula tobacco can effectively improve the quality of cigarettes.

A Comparative Study on Extraction Methods of Total RNA from Leaves of Acer truncatum ‘Luhong No.1’

Leaves of Acer truncatum ＇ Luhong No. 1 ＇ contain large amounts of polysaccharides and polyphenols, which seriously affect extraction yield and quality of total RNA. In order to explore the appropriate total RNA extraction method, total RNA was extracted from leaves of A. truncatum ＇ Luhong No. 1 ＇ with three methods, including kit method, Trizol method and modified CTAB method. The results showed that ODE6o/OD2so and OD^o/OD2ao ratios of total RNA extracted from leaves of A. truncatum ＇ Luhong No. 1 ＇ with kit method were higher than 1.8, with a general yield and certain level of DNA contamination ; OD^o/OD~ and OD^o/OD^o ratios of total RNA extracted with Trizol method were about 1.5, with the lowest yield; OD260/OD280 and OD260/OD230 ratios of total RNA extracted with modified CTAB method were about 2.0, with the highest yield and distinct eleetrophoresis patterns. The results demonstrated that total RNA extracted with modified CTAB method exhibited high yield and purity, which could meet the demands of subsequent molecular biology research. Thus, modified CTAB method is the appro- oriate method for extracting total RNA from leaves of A. truncatum ＇ Luhong No. 1 ＇

A Comparative Study on Three Methods for the Extraction of Total RNA from Pinus bungeana

[Objective] The aim of this study was to compare three RNA extraction methods and thus find out the suitable one for isolating intact and high quality total RNA from Pinus bungeana.[Method] Employing three extraction methods of Trizol,RNeasy Mini Kit,LiCl precipitation,total RNAs of P.bungeana were extracted from pine leaf samples,and their integrity and purity were detected via agarose gel electrophoresis and spectrophotometry for a comparative study.[Result] Among the three extraction methods,LiCl precipitation method demonstrated higher yield and better integrity of total extracted RNA,with OD260/OD280 ratio between 1.8-2.0 and clear 28 S and 18 S bands in electrophoresis pattern.[Conclusion] LiCl precipitation method could be used to extract highly pure and intact total RNA from P.bungeana.

Comparative Study on the Extraction of Total RNA from Different Tissue Parts of Cone Snail(Conus geographus)

[Objectives] This study was conducted to compare the quality of total RNA extracted from different tissues of cone snail( Conus geographus).[Methods] The total RNA of four different tissues of cone snail,venom gland,venom tube,salivary gland and tooth gland was extracted by the Trizol method. The total RNA of cone snail was detected by agarose gel electrophoresis,NanoDrop^(TM) 2000 spectrophotometer and Aligent 2100 biological analyzer. [Results]The total RNA extracted from different tissues of cone snail showed clear band,and thus had similar concentrations and purity,and the highest yield was obtained from venom tube. [Conclusions] The total RNA extracted from different tissue parts of cone snail could meet the basic requirements of molecular biology,and its venom tube was the best tissue part for extracting total RNA,which lays a foundation for molecular biology research such as high-throughput transcriptome sequencing of cone snail.

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（15.68～35.2 μg.g-1）低于SDS/酸酚法提取RNA的含量 （46.5～51.9 μg.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.

血清HCV RNA不同提取方法的对比研究

目的:探讨不同方法提取丙型肝炎患者血清HCVRNA,比较每种方法提取的RNA用于RT-PCR荧光定量检测的敏感度。方法:分别采用异硫氰酸胍-酚氯仿法、蛋白酶K-酚氯仿法、固相吸附法、柱式总RNA抽提法提取7例疑似HCV感染者血清标本中的HCVRNA,采用实时荧光定量PCR法进行检测。结果:采用异硫氰酸胍-酚氯仿法、蛋白酶K-酚氯仿法提取HCVRNA用于核酸检测有漏检现象,而采用固相吸附法和柱式总RNA抽提法,敏感度均可达到100%。结论采用固相吸附法和柱式总RNA抽提法提取血清HCVRNA具有较高的敏感度,在实时荧光定量PCR法检测中具有重要的临床价值。

改良CTAB法提取不同作物总RNA技术研究

为提高操作效率,本研究开发出一种简洁有效的作物总RNA提取方法。研究以油菜、棉花、花生、小麦、马铃薯、玉米、大豆、西葫芦等作物的不同组织为试材,通过CTAB法、改良CTAB法及TanSzol Reagent试剂盒法提取总RNA,检测结果发现,经过改良后的CTAB法提取的总RNA质量较高。在油菜花瓣、小麦、花生、西葫芦叶片中,该方法获得了较高的总RNA得率,分别为24.5、23.7、26.4、17.35μg/mL;OD260/280和OD260/230值分别为1.90、1.73、1.72、1.80和2.10、1.50、2.01、1.70。综合比较以上3种方法,改良后的CTAB法适用于油菜、小麦、花生、西葫芦等作物的总RNA提取,为后续分子生物学检测奠定了基础。

基于LiCl法优化酿酒葡萄叶片的RNA提取

为获得纯度高、质量好的酿酒葡萄植株叶片RNA以用于荧光实时定量分析,为后续以酿酒葡萄叶片RNA为材料研究相关转录水平的调控提供技术保障,以经典酿酒葡萄赤霞珠叶片为试验材料,比较分析了重蒸酚法、Trizol法、改良的十六烷基三甲基溴化铵(CTAB)法和LiCl法(基于改良的SDS法)4种方法对酿酒葡萄叶片RNA提取的影响。结果表明,重蒸酚法提取RNA浓度较低,完整性较差,存在部分降解;Trizol法提取的RNA则有部分DNA污染;改良CTAB法提取浓度较高,但蛋白和DNA污染严重;LiCl法提取结果显示,28S的条带亮度是18S的2倍,条带无拖尾,完整性较好,存在DNA污染和少量的蛋白污染。进一步在LiCl法基础上利用醋酸钠、DNAase消化、苯酚抽提法进一步优化,并采用荧光实时定量验证,结果表明,醋酸钠的加入RNA质量无明显改善;然而,经氯仿和水饱和酚(体积比1∶1)抽提2次,氯仿抽提1次,氯仿和水饱和酚(体积比1∶1)抽提1次,并经DNAase进行消化,最终提取出质量浓度为173.611μg/mL、A_(260/280)为1.803纯度高、完整性较好且无蛋白和DNA污染的葡萄叶片总RNA;经实时荧光定量PCR进一步验证,该方法提取的RNA经反转录后获得较好的扩增曲线以及融解曲线,并能够对相关基因进行定量分析。

反转录酶在微小RNA实时荧光定量聚合酶链反应中的作用

目的以微小RNA(miRNA)作为检测样本,考察影响实时荧光定量聚合酶链反应(RT-qPCR)检测结果的关键点,并筛选出最优的检测方法。方法采用3种miRNA提取方法(分别使用EasyPure^(■)miRNA提取试剂盒、miRNA提取试剂盒、TransZol Up Plus RNA提取试剂盒)和3种反转录方法(分别使用TransScript^(■)第一链cDNA反转录试剂盒、Evo M-MLV反转录试剂盒、miRNA第一链cDNA合成试剂盒)处理大鼠纹状体脑区,检测miRNA与c DNA的质量和效率,并使用常规RT-qPCR检测miRNA水平,比较不同方法所得结果。结果3种RNA提取方法得到的miRNA质量和效率比较差异均有统计学意义,其中方法2的效果较好,但方法1、2、3的RT-qPCR结果比较差异无统计学意义(miR-132:25.91±9.79、25.26±10.25、27.28±7.39,miR-U6:27.98±11.25、25.98±9.78、29.62±9.65,均P>0.05);3种反转录方法所得实验结果比较差异有统计学意义,方法3所得结果明显低于方法1、方法2(miR-132:16.53±3.17比35.20±1.06、31.42±2.95,miR-U6:16.63±1.73比36.06±2.57、35.59±1.54,均P<0.05),主要影响RT-qPCR的扩增效率和扩增特异性。结论使用能高效富集约18 nt大小RNA的提取方法和在miRNA的3’末端加多聚A尾(Poly A)的反转录酶,可以得到更可靠的RT-qPCR结果。

烟草废弃物提取物的双水相萃取及其化妆品功效研究

采用双水相萃取法制备烟草废弃物提取物,探究不同萃取条件对其中上相多酚得率的影响,并探讨其抗氧化活性、紫外吸收特性和细胞毒性。结果表明:当料液质量比为1∶60,搅拌时间为30 min,(NH4)2SO4质量分数为23.3%,乙醇质量分数为23.5%时,上相多酚得率最高,为48.59 mg/g;该萃取条件下,烟草废弃物提取物具有较好的抗氧化活性,当质量浓度为2 mg/mL时,对DPPH自由基清除率达98%,对ABTS自由基清除率达78%;当提取物质量浓度为0.5 mg/mL时,具有较好的紫外吸收特性,有一定的光防护功效;当提取物质量浓度在0~1.2 mg/mL范围内时,对Hacat细胞无细胞毒性,有望成为天然无刺激的功效型化妆品原料。

一种适用范围广的总RNA提取方法

介绍一种RNA提取方法,该方法以SDS、氯仿和Tris苯酚为主要提取试剂,以LiC l和乙醇为RNA沉淀试剂。分别以柽柳(木本植物)、星星草(草本植物)、天牛(昆虫)、酿酒酵母和白腐菌(真菌)为RNA提取材料,用该方法成功地提取出了它们的总RNA。获得的RNA条带清晰,A260/A280在1.8以上。通过对LiC l和乙醇沉淀RNA的效果分析表明,该方法可在10 m in内完全沉淀RNA,同时也可以同时获得纯度较高的DNA。提取的RNA质量可满足cDNA文库构建,基因芯片探针标定和RT-PCR等对RNA质量要求较高的分子生物学操作,说明这是一种应用范围广的RNA提取方法。

一种改良的提取草莓属叶片总RNA的方法

探索一种新的适合草莓属(Fragariaspp.)组培苗、大田苗叶片总RNA的快速高效提取方法。试验以草莓叶片为材料,用改进的CTAB法对其RNA进行提取,并对提取的RNA进行了电泳检测、含量测定和RT-PCR检测。结果表明:用该RNA提取方法提取的RNA具有28 S rRNA、18 S rRNA和5.8 S rRNA 3条清晰的条带,且无降解。OD260/OD280在1.83至2.14之间,具有较高的纯度,且含量较高。用该RNA提取方法提取的RNA逆转录成cDNA,经PCR扩增出现清晰的条带,说明该RNA提取方法提取的RNA可以满足进一步分子生物学试验的要求。

百合叶片总RNA提取方法比较及优化

为筛选出适合百合叶片RNA提取方法,以铁炮百合‘白天堂’组培苗叶片为材料,比较了改进的CTAB、SDS及传统的Trizol、CTAB、SDS方法提取RNA的效果。结果表明:不论传统及改进的SDS法和Trizol法均不能有效去除百合叶片组织中的多糖、蛋白等杂质,传统CTAB法提取RNA存在DNA污染。针对百合叶片组织富含多糖多酚的特点,改进了CTAB法,采用醋酸钠及无水乙醇去除多糖,酚/氯仿反复抽提去除蛋白,DNaseⅠ去除DNA,LiCl有效沉淀RNA。采用此方法提取的RNA条带清晰,经紫外光谱分析D260/D280比值为2.0,D260/D230为2.1,电泳28S、18S条带清晰,比值约为1.5。RT-PCR结果表明,改进的CTAB法提取的总RNA可直接用于分子克隆和基因表达分析等分子生物学实验。

Trizol试剂法快速高效提取3种作物不同组织总RNA

【目的】建立适用于禾本科作物不同组织总RNA的快速、高效提取方法,为进行后续的分子生物学研究奠定基础。【方法】采用Trizol试剂法,通过改变抽提上清液吸取量、沉淀方法及沉淀漂洗次数,对甘蔗、玉米和水稻的叶片、茎尖和根尖的总RNA提取方法进行优化。【结果】与吸取0.4mL上清液相比,吸取0.2mL上清液的总RNA OD260/OD280、OD260/OD230值分别在1.95～2.00和2.11～2.44,所获得的总RNA纯度较高。沉淀漂洗两次的OD260/OD230值(2.11～2.47)明显高于沉淀漂洗1次的总RNA OD260/OD230值(1.01～1.61),总RNA纯度较高。经异丙醇沉淀所得的各材料的总RNA28S、18S和5S谱带清晰,无拖尾现象,总RNA完整性较好;LiCl沉淀法的RNA条带较模糊粘连,5S条带明显弥散,总RNA产率相对较低。以Trizol-异丙醇法提取的甘蔗总RNA为模板进行GAPDH基因片段扩增,所获甘蔗的叶片、茎尖和根尖的GAPDH基因表达丰度很强,无特异扩增,目的片段长度为153bp。【结论】改良Trizol-异丙醇法提取甘蔗、玉米和水稻不同组织的总RNA带型清晰、完整性好、纯度和产率高,适用于快速、高效提取禾本科植物不同组织总RNA。

烟草总RNA高效提取方法研究

作为一个模式植物,烟草在植物分子生物学研究中发挥着重要的作用。总RNA的提取质量,对于基因表达、基因克隆、cDNA文库建立、RNA原位杂交以及转基因材料鉴定等分子生物学基础研究至关重要。尤其在进行基因克隆及cDNA文库时,只有高质量的RNA,才能保证cDNA第一链合成的完整性。由于烟草中糖类、蛋白质及次生代谢物的含量较高,因此,在应用TRIzolRNA提取试剂盒进行烟草总RNA提取时,所提取的RNA产物中的蛋白质及多糖等杂质含量偏高。因此,针对这一问题,我们对TRIzol法进行了改进,并获得了高质量的烟草总RNA。经琼脂糖电泳检测,利用该方法提取的烟草总RNA,条带清晰、无拖尾现象,28S与18S的比例约为2:1。紫外吸收值的测定表明,所提取的烟草总RNA的A260/A280的值基本达到了1.9以上,说明所提取的RNA的质量较高,可广泛应用于基因的克隆及基因的表达研究。

一种简便的同步提取烟草叶片DNA和总RNA的方法

以野生型和转几丁质酶烟草株系叶片为材料,采用改进的CTAB法对其DNA和总RNA进行同步提取和DNA中RNA的消化以及总RNA中DNA的消化,并分别进行了电泳检测、含量测定和相应的PCR和RT-PCR检测。结果表明:用该法提取的DNA条带清晰、无降解,所提取的DNA浓度在109.20～245.46μg/mL,OD_(260)/OD_(280)在1.83～1.94。以提取的DNA为模板,PCR能够扩增出清晰的目的条带。RNA具有5.8SrRNA、18S rRNA和28S rRNA 3条清晰的条带,且无降解。RNA浓度在355.57～570.13μg/mL,OD_(260)/OD_(280)在1.84～2.04,OD_(260)/OD_(230)在2.06～2.33。RNA逆转录成cDNA,经PCR扩增出清晰的看家基因和目的基因条带。

孝顺竹RNA提取方法研究

提取纯度高、完整性好、不含DNA或其它杂质的总RNA是进行Northern杂交、cDNA合成、cDNA文库构建、RT-PCR及mRNA差异显示等分子生物学研究的基础.目前提取植物RNA方法有多种,这些方法设计旨在RNA提取过程中清除多糖、多酚等杂质污染.在提取高质量RNA的过程中,植物尤其木本植物通常含有大量的酚类、多糖和其他一些水溶性化合物与RNA共沉淀,影响了RNA功能[1].目前应用较多和较广的有5种方法,商业试剂盒TRIzol,以异硫氰酸胍为代表异硫氰酸酸胍法[1,2]、以阳离子去污剂CTAB代表CTAB法[3,4,5]、热硼酸盐法[6,7],和阴离子去污剂SDS为代表SDS法[8～11]等.结合蛋白质、多糖和多酚去除,以及RNA沉淀形成的这些方法,往往要依据不同植物进行适当改进.

改良Trizol法提取香蕉叶片总RNA

针对香蕉组织中多糖、多酚等次生物质含量高的特点,采用改良Trizol法,成功地从香蕉叶片中提取了总RNA。所提取的总RNA A260/A280和A260/A230值分别为1.91和2.10。电泳检测显示28 s、18 s条带完整清晰,以此RNA为模板进行RT-PCR,结果成功得到目标特异条带。试验结果表明:改良Trizol法具有方便快捷、完整性好、受多糖多酚影响较少以及无DNA和蛋白质污染等优点,对具有香蕉叶片类似成分的其他作物的RNA提取具有一定的借鉴意义。

烟草叶片总RNA提取方法的比较

[目的]研究提取烟草叶片总RNA的最佳方法。[方法]利用CTAB法、酚-SDS法和Trizol法等从烟草叶片中提取总RNA。[结果]通过紫外分光光度法和凝胶电泳检测,结果表明,CTAB法、Trizol法、RNAiso法均能得到质量较高的RNA。RNAiso法提取RNA操作步骤简单,所提取的RNA纯度好、质量高且无污染,可以进行下一步的分子生物学研究。[结论]RNAiso法最适用于烟草叶片总RNA的提取。