非洲菊花瓣总RNA提取方法的改进

利用异硫氰酸胍一步法和植物总RNA提取试剂盒（RNeasy Plant Mini Kit）提取非洲菊（Gerbera hybrida）花瓣中总RNA时存在多糖的干扰。实验中利用异硫氰酸胍一步法提取总RNA,在RNA沉淀后,再次加入适量变性液,冻融2次,再加热至45 ℃使RNA完全溶解;试剂盒提取时,适当延长各提取步骤的离心时间,并增加DEPC H2O溶解RNA的时间。通过以上操作方法的改进可排除多糖的干扰,得到高质量的总RNA。为进一步利用Northern杂交技术研究非洲菊中花色素苷基因的表达,进行花色改良提供了方便、有效的实验手段。     

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。     

几种提取白木香茎干总RNA方法的比较

目的:通过比较多种RNA提取方法,确定白木香茎干RNA提取的有效手段,为白木香伤害形成沉香药材的分子研究奠定基础。方法:取2年生白木香茎,分别用CTAB法、TRIzol法、异硫氰酸胍-SDS法、Qiagen试剂盒和Nor gen试剂盒法提取总RNA,通过琼脂糖凝胶电泳和紫外分光光度法测定其产量与质量,并通过cDNA合成及小样本克隆测序检查RNA的可用性。结果:异硫氰酸胍-SDS法及Norgen试剂盒法提出的RNA条带清晰,完整性好;D260nm/D280nm值可达2.0左右,纯度较高,其产量足够应用于cDNA合成;其他方法不能提出RNA。结论:异硫氰酸胍-SDS法及Norgen试剂盒法可用于白木香茎干RNA提取。     

藤茶叶片总 RNA 的提取及苯丙氨酸解氨酶基因（pal）片段的克隆

藤茶叶片富含多酚、多糖以及黄酮类化合物,严重干扰了其总RNA的提取。实验在裂解前先用70%丙酮对藤茶叶片冷冻研磨材料进行多次抽提,然后采用试剂盒提取法、Trizol法和异硫氰酸胍法提取总RNA。结果表明,经丙酮预处理后,上述3种方法均能从藤茶叶片中成功提取到高质量的RNA,其完整性好,纯度高,D260 nm/D280 nm值介于1.8~2.0之间,产率为22.74~33.66μg/g。以提取的总RNA逆转录的c DNA为模板进行RT-PCR,克隆到一条长度为817 bp的pal片段。经Blast比对分析,该基因片段与葡萄、茶树等物种的pal之间具有较高的同源性。     

几种提取RNA的方法

以肝脏组织和NDV感染的鸡胚尿囊液为例,简单介绍了几种提取细胞或组织液中总RNA及mRNA的方法－SOD－蛋白酶K法、异硫氰酸胍法、loigo(dT）纤维素亲和层析法。SDS－蛋白酶K法提取RNA经济可靠,但纯度稍低;异硫氰酸胍法适于提取细胞中的总RNA,纯度较高;利用ligo(dT)能与mRNA poly（A^ )尾结合的特性提取组织细胞中的mRNA.     

改良异硫氰酸胍法提取玛咖(Maca)叶片中总RNA研究

为了获得质量较高的玛咖总RNA,对其总RNA的提取进行了研究。以玛咖无菌苗叶片为材料,通过改良的异硫氰酸胍法提取其总RNA。结果:所提取的总RNA纯度高、完整性好,电泳条带清晰,OD260/OD280值达到1.90±0.03,总RNA的得率达到252.57±1.12μg/g。表明改良的异硫氰酸胍法适合于玛咖叶片中RNA的提取,所提取的总RNA纯度高、完整性好,为进一步的玛咖分子生物学研究打下良好基础。     

提取高质量人参RNA的方法研究

针对人参组织多酚、多糖类物质含量较高的特点,比较了改良的异硫氰酸胍法、改良的CTAB法和改良的Trizol法等3种不同的RNA提取方法.3种改良的方法均能从人参组织中提取到总RNA.其中改良的Trizol法能有效地抑制酚类物质和多糖对总RNA提取的影响,能从成熟的叶片中获得高质量、完整性好的总RNA,每克新鲜组织RNA产量在90～120!g之间,电泳分析,28SrRNA亮度约为18SrRNA的2倍,A260/A280介于1.8～2.0之间.用改良的Trizol法分离的RNA,已成功进行了RT-PCR及人参叶cDNA文库构建等研究.     

山药组织总RNA提取方法的比较与分析

采用异硫氰酸胍-巯基乙醇联合变性法、Trizol法和CTAB-LiCl法等3种方法,分别对山药叶片和块茎进行总RNA提取效果进行了比较.结果表明,Trizoi法难以提取RNA,异硫氰酸胍-巯基乙醇联合变性法提取RNA效果不理想,存在DNA污染,这2种方法不适合于富含多糖类物质的山药组织总RNA的提取;CTAB-LiCl法提取叶片和块茎组织总RNA质量高、完整性好、成功率高,可作为山药类植物总RNA提取的首选方法.     

草坪草总RNA几种提取方法的比较

分别采用DEPC水法、TRIzol法、CTAB-LiCl沉淀法、SDS-酚法、SDS提取液抽提法以及异硫氰酸胍法等6种方法提取草坪草总RNA,通过检测对各种方法的提取效果进行比较分析。结果表明,DEPC水法是最经济、操作最简单的一种方法,并且提取的RNA质量较好、可靠性高、易于大量制备,是提取草坪草RNA比较理想的方法之一,建议使用该方法。利用TRIzol法提取到的28S rRNA亮度约为18S rRNA的两倍,效果较好,操作简单,符合分子生物学实验要求。CTAB-LiCl沉淀法、SDS-酚法、SDS提取液抽提法也获得较高质量的RNA,但存在不同程度的DNA污染。异硫氰酸胍法提取RNA有明显的蛋白质污染,且RNA部分降解,此种方法不予采用。     

脂肪组织RNA提取方法的改进

本试验以猪脂肪组织为材料,应用TRIzol、V-gene、H.Q＆.Q三种总RNA提取试剂盒分别提取脂肪组织RNA,发现直接按照试剂盒说明书操作提取的RNA效果不理想.通过反复试验,分别改进了提取过程中的部分操作步骤,结果表明：三种试剂盒操作方法改进后与改进前相比,提取RNA的纯度和得率表现为差异极显著（P＜0.01）;三种试剂盒操作方法改进后提取RNA的纯度差异不显著（P＞0.05）,但在得率方面,Trizol比其它两种高,表现为差异极显著（P＜0.01）.三种试剂盒操作方法改进后提取的RNA经RT-PCR扩增猪β-actin基因,电泳结果显示,扩增条带清晰、特异,说明提取的RNA完全符合后续的分子生物学实验要求.     

Recovery comparison of two virus concentration methods from wastewater using cell culture and real-time PCR

Enteric viruses are shed in the feces and may be present in environmental waters. Their detection in wastewater, even at low concentration, is a major challenge. In this study, recoveries of Echovirus 7 (EV7), virions and RNA in wastewater, using virus concentration methods were determined to evaluate the detection of infectious viruses and the possibility of recovering viral genomes. Two virus concentration methods, PEG precipitation method and two-phase separation method, were applied to recovery experiments of EV7-virions from wastewater, in parallel with recovery experiments of EV7 RNA. The titration of EV7 virions was carried out by cell culture using human rhabdomyosarcoma tumor tissue and the EV7 RNA quantification was performed by real-time PCR. The mean recovery yields of EV7 virions using the PEG precipitation method and the two-phase separation method were 78.5?±?10.99 and 83.1?±?0.28?%, respectively. Besides, EV7 RNA recoveries obtained using the PEG precipitation method were four times higher than those using the two-phase separation method. According to our results, the two methods enable to concentrate both infectious viruses and viral genomes. Moreover, considering the protocol time and cost together with the ratio of the EV7 virion recovery to the EV7 RNA recovery, the two-phase separation method (83.1/2.71?%, or 30.6) seems to be more appropriate for selective concentration of viral virions than the PEG precipitation method (78.5/10.33?%, or 7.6).     

从水稻种胚中提取RNA的新方法

介绍了一种全新、简单的提取水稻种胚RNA的CTAB-LiCl提取法,可在不用液氮的室温下有效地排除剥取的种胚中大量带有的多糖和本身脂质的干扰,所得RNA样品经紫外分光光度计和琼脂糖凝胶电泳分析证明具有较高的纯度和完整性,并可进一步满足RT-PCR和Northern 印迹的实验需要,适用于富含多糖和脂质的植物组织RNA的提取。 Abstract:A novel and simple CTAB-LiCl-based extraction method for high-quality and total RNA of rice embryo samples is developed.This method can efficiently eliminate the interference of polysaccharide and lipids rich in rice（Oryza sativa L.）embryo obtained under room temperature without using liquid nitrogen.The results of ultraviolet spectrophotometer and agarose gel electrophoresis analysis show that the obtained RNA has no obvious degradation and a good purity sufficient for further RT-PCR and RNA gel blotting.Therefore,it is also especially useful for the RNA extraction of plant material plenty of polysaccharide and lipids.     

生物大分子分离技术:过去、现状和未来

生物大分子包括多肽、酶、蛋白质、核酸(DNA和RNA)以及多糖等。生物大分子分离技术是生命科学研究中的关键技术之一。当前,各学科之间的交叉渗透为生物大分子分离技术的发展提供了更多的契机。对以沉淀、透析、超滤和溶剂萃取为代表的传统分离技术,以及色谱,电泳等现代分离技术的发展概况、原理、特点及应用进行了综述。并结合生命科学的发展现状,展望了生物大分子分离技术的发展前景。     

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。     

Determination of Antibody to Pneumococcal Polysaccharides with Chromic Chloride-Treated Human Red Blood Cells and Indirect Hemagglutination

A method is described for the quantitation of serum antibody to type-specific pneumococcal polysaccharide. The method uses highly purified pneumococcal polysaccharide coated onto human O+ red blood cells by the chromic chloride technique. Each of 14 pneumococcal polysaccharide types was individually coated onto red blood cells and used to determine the antibody response following primary immunization. The method was found to be sensitive, detecting antibody titer increases of several hundred to a thousand-fold. The presence of high preimmunization antibody titers did not obscure the detection of antibody titer increases. The method detected antibody of both the immunoglobulin M and immunoglobulin G class when quantitated after ultracentrifugation and sucrose density gradient separation. By using serum samples obtained from volunteers immunized with a single pneumococcal polysaccharide, the method was standardized resulting in an ability to compare samples taken at different times and obtained from different sources. The method appears to be simple, reproducible, and inexpensive and can be utilized to determine the antibody response following immunization in large population studies.     

Reversed-phase ion-pair liquid chromatography analysis and purification of small interfering RNA

Small interfering RNA (siRNA)-induced gene silencing shows great promise in genomic research and therapeutic applications. siRNA duplexes are typically assembled from complementary synthetic oligonucleotides. High-purity single-stranded species are required for in vivo applications. Methods for separation, characterization, and purification of short RNA strands have been developed based on reversed-phase ion-pair liquid chromatography. The purification strategies were developed for both single-stranded and duplex RNA species. The method of duplex purification uses on-column annealing of complementary RNA strands, followed by separation of the target duplex from truncated duplexes and single-stranded RNA forms. The proposed method significantly reduces the purification time of synthetic siRNA.     

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。     

淡紫拟青霉胞外多糖的分离、纯化及结构分析

淡紫拟青霉NH-PL-03菌株的胞外多糖粗提物对枯萎病病原菌-尖孢镰刀菌具有较好的抑制效果,文中对淡紫拟青霉胞外多糖进行了分离纯化和结构分析,以期为其构效关系研究奠定基础。采用乙醇沉淀法从淡紫拟青霉发酵液中提取粗多糖,经Sevage法脱蛋白后,过Superdex-G75凝胶层析柱分离得到胞外多糖EP-1。紫外分光法和Sephacryl S-200 HR凝胶层析柱检测EP-1为均一多糖,Sephacryl S-200柱层析测得EP-1的分子量为35.2 kDa,完全酸水解后纸层析检测EP-1的单糖组成中仅有葡萄糖,红外光谱、高碘酸氧化和Smith降解结果表明EP-1的化学结构是以β-(1,3)糖苷键连接而成的无分枝的葡聚糖。刚果红络合试验表明EP-1在稀的碱溶液中以3股螺旋构象存在。     

Analysis of partially methyl-esterified galacturonic acid oligomers by capillary electrophoresis.

Recent progress in the determination of the intramolecular distribution of methyl-esterified residues in pectic substrates has been made using a fragmentation approach in which endopolygalacturonase is used to digest the polysaccharide and its subsequent (methyl-ester sequence-dependent) digest pattern is determined. This has been facilitated by the separation of partially methyl-esterified enzyme digest fragments using high-performance anion-exchange chromatography at pH 5. Here we demonstrate that capillary electrophoresis can be used as an additional method for separation and quantification of such digest patterns. The technique offers improvements in speed and economy of materials and a straightforward quantification procedure.