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。     

从动物组织提取高纯度总RNA方法的改进及应用

从动物组织中提取总RNA是现代分子生物学研究中经常使用的重要实验技术,按常规方法获得的总RNA产品中常伴有大分子量染色体DNA污染。为此,我们摸索出了保证RNA制品纯度、质量和产量的DNA酶消化最佳反应条件。利用改良后的方法提取了小鼠脏器组织总RNA,进行了新基因mPC-1在小鼠脏器中表达的组织分布研究。     

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。     

一种适于提取荔枝花与幼果组织总RNA的方法

介绍了一种从荔枝花与幼果组织中提取高质量和较高产量的总RNA的方法,该方法提取的总RNA可以满足构建cDNA文库、开展RT-PCR、Northern杂交分析、基因表达差示分析等方面研究的要求。     

外来入侵植物紫茎泽兰不同组织RNA提取方法

从植物组织中提取高质量的RNA是进行cDNA文库构建等分子生物学研究的前提。在苯酚法的基础上,改进并得到了一种适合紫茎泽兰根、茎、叶总RNA快速提取的方法,消除了蛋白质、DNA、多糖等的污染。该方法提取的紫茎泽兰不同组织总RNA纯度高、完整性好,可用于RT-PCR、cDNA文库构建、Northern杂交等分子生物学实验,而且简单、经济、重复性好,适合于多种植物组织总RNA的提取。Northern杂交表明F3’H基因在紫茎泽兰的根、茎、叶等组织中广泛存在,但在叶中的表达量最高,在根中的表达量最低。     

应用非伤害性取样提取番鸭毛囊组织总RNA

目的:寻求一种从番鸭毛囊组织中高效提取总RNA的方法。方法:探讨了伤害性取样(剪切皮肤毛囊)、非伤害性取样(直接拔取毛囊)2种不同毛囊取样法对总RNA提取质量的影响,并对常用RNA提取方法TRIzol法中研磨和组织匀浆步骤细节稍加改进,琼脂糖电泳检测总RNA质量。结果:2种毛囊取样方法均能提取出高质量的总RNA,其28S、18S和5S条带清晰可见,无DNA污染。结论:非伤害性取样法可作为番鸭毛囊组织总RNA提取的适用取样方法。     

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.     

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。     

柠檬总RNA提取方法研究

为了获得高质量的柠檬RNA,本研究从操作时间、成本、所得RNA的纯度与浓度等方面比较了5种方法提取香水柠檬不同组织总RNA的效果,并筛选最适合提取柠檬组织样品RNA的方法。结果表明:改良的RNA试剂盒提取法效果最好。该方法极适合提取香水柠檬总RNA。利用此方法可以成功提取白花柠檬、广西土柠檬和金柑的总RNA,也可以提取高质量的芒果总RNA。     

荒漠甲虫总RNA提取方法的比较与分析

目的:昆虫总RNA由于其自身结构特点,存在与动物植物完全不同的电泳条带,本文通过对比探索光滑鳖甲总RNA提取的最优方案。方法:实验采用5龄光滑鳖甲幼虫,利用TRIzol、CTAB、热酚法、及BioTeKe Kit、TianGen Kit试剂盒提取方法提取光滑鳖甲总RNA。结果:(1)所采用的实验方法提取的光滑鳖甲总RNA均呈现出与植物、哺乳动物血细胞、哺乳动物组织样总RNA不同的条带,28S弱于18S条带亮度;(2)TRIzol法、CTAB法、改进热酚法所得光滑鳖甲总RNA条带清晰、完整,D260/D280值为1.89～1.98,D260/D230值为1.94～2.09,纯度较好;(3)TRIzol法RNA提取得率为304.3～365.4μg/g,BioTeKe Kit RNA提取得率为73.38～128.22μg/g。结论:综合所有参数,所选取的方法中,TRIzol法可获得高质量、高产量的光滑鳖甲总RNA,可用于RT-PCR、文库构建等高要求RNA的提取。     

The single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction: twenty-something years on

Since its introduction, the 'single-step' method has become widely used for isolating total RNA from biological samples of different sources. The principle at the basis of the method is that RNA is separated from DNA after extraction with an acidic solution containing guanidinium thiocyanate, sodium acetate, phenol and chloroform, followed by centrifugation. Under acidic conditions, total RNA remains in the upper aqueous phase, while most of DNA and proteins remain either in the interphase or in the lower organic phase. Total RNA is then recovered by precipitation with isopropanol and can be used for several applications. The original protocol, enabling the isolation of RNA from cells and tissues in less than 4 hours, greatly advanced the analysis of gene expression in plant and animal models as well as in pathological samples, as demonstrated by the overwhelming number of citations the paper gained over 20 years.     

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.     

Detection of mRNA degradation intermediates in tissues using the 3'-end poly(A)-tailing polymerase chain reaction method

It has become increasingly clear that mRNA stability is an important determinant of mRNA abundance in virtually all organisms. Although our understanding of prokaryotic lower eukaryotic mRNA stability mechanisms has progressed considerably, little is known about mammalian mRNA stability mechanisms, particularly at the tissue and animal levels. This is due largely to the lack of suitable methods to approach the problem. In this study, we have developed and refined the 3'-end poly(A)-tailing polymerase chain reaction (PCR) method to detect degradation intermediates in vivo. Using an in vitro transcribed RNA as a template, we found that the method could be used to detect a homogeneous pool of RNA down to 0.1 ng. The addition of 10 microg of total RNA from tissues decreased the sensitivity limit to 4 ng. Detection limits of the technique were determined precisely by varying the concentrations of in vitro transcribed RNA in a constant amount of total RNA and varying the concentration of total RNA while maintaining a constant amount of in vitro transcribed RNA. Our overall results showed that the poly(A)-tailing PCR method could be used to detect specific RNA species of approximately 1000 nt in a pool of heterogeneous RNA in the range of 1 in 2500 to 1 in 10,000. To our knowledge, this is the most sensitive method to date for identifying mRNA degradation intermediates. Employing sense strand gene-specific primers in this method, we have discovered the class II and class III P-glycoprotein (Pgp) mRNA degradation intermediates in normal rat tissues. This method should serve as an additional tool to help us understand mRNA decay mechanisms in tissues and at animal levels.     

A gastrin gene is expressed in both porcine pituitary and antral mucosal tissues.

By employing S1 nuclease mapping of total RNA isolated from porcine cerebral cortex, cerebellum, hypothalamus, pituitary, kidney, liver, pancreas, intestine, and antral mucosa, we have investigated gastrin gene expression in these tissues. Our results show that a gastrin gene is expressed only in the antral mucosal and pituitary tissues. Based on the amount of gastrin specific probe protected from S1 nuclease digestion in the presence of a given weight of total RNA, the amount of gastrin mRNA present in pituitary is approximately 330 times lower than in antral mucosa. These findings help establish the tissue distribution of gastrin gene expression.