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 .     

一种提取小型昆虫总RNA的有效方法

实验采用SDS,乙酸钾(KAc)等常规化学试剂从蚜虫等小型昆虫中获得完整且纯度较高的RNA。进一步的实验证明提取的RNA可用于RT-PCR以及cDNA文库的建立等。该方法简捷、相对安全和快速、提取效率较高,适合于中小型昆虫RNA的提取。     

简便、快速提取高质量的RNA分子

MethodsforHandy,RapidIsolationofHighQualityRNAbyGuanadiumThioeyauateDuJianYuanYanhuaMaShenglinDongZhiwei(BeijingInstituteforCancerResearchBeijing100034)硫氰胍是一种有效的蛋白变性剂。早在1979年,Chirgwin等就利用氯化铯／硫氰胍超离心技术成功地从RNA酶富集的胰脏组织中提取出未降解的RNA分子[2],从而使它成为抑制RNA酶的首选药物并得到广泛使用,但受到超速离心设备的限制。1983年,Cathala报道了氯化锂／硫氰胍RNA提取法[1]。该方法操作简便,获得的RNA质量很高,但所需时间较长。为了能在短时间内更快…     

槟榔黄化病组织RNA提取方法的比较

为了从发生黄化病的槟榔茎、叶、花中提取到完整的RNA,采用了CTAB法、Trizol法和异硫氰酸胍法提取槟榔黄化病组织RNA。从RNA的完整性、产率和纯度方面对这几种方法进行了比较,结果表明,异硫氰酸胍法所得RNA完整性较好,条带清晰无明显降解,OD260/OD280介于1.8-2.0之间,RNA得率较高,为120μg/g以上,并能成功进行反转录制备cDNA,表明该RNA可以进行后续分子生物学操作。     

五种不同组织结构特点昆虫的总RNA提取效率比较

【目的】比较RNAeasy抽提试剂盒法和Trizol提取法对不同种昆虫总RNA提取的效率,为不同种昆虫总RNA提取方法选择提供参考。【方法】采用RNAeasy抽提试剂盒法和Trizol提取法分别对九香虫Aspongopus chinensis Dallas、白背飞虱Sogatella furcifera Horváth、中华蜜蜂Apis cerana cerana、黄蜻Pantala flavescens和蒿金叶甲Chrysolina aurichalcea进行总RNA提取,通过紫外分光光度计测定总RNA吸光度(OD)值,利用琼脂糖凝胶电泳检测等方式对RNA质量进行评估。【结果】2种提取方法提取不同昆虫总RNA中,Trizol法提取白背飞虱(同翅目)、蒿金叶甲(鞘翅目)和九香虫(半翅目)的总RNA浓度较高,但纯度较低。电泳结果显示总RNA有降解,完整性较差,RNAeasy抽提试剂盒法提取黄蜻(蜻蜓目)和中华蜜蜂(膜翅目)的浓度较低,纯度较高,电泳结果显示完整性较好。【结论】Trizol法更适合提取小型或几丁质含量高的昆虫,RNA抽提试剂盒法更适合几丁质含量较少、腹软且有大量体液的昆虫总RNA提取。     

甜叶菊RNA分离方法研究

甜叶菊组织中酚类、萜类和多糖等代谢产物含量较高,RNA难分离,易降解,使用现有的方法提取甜叶菊组织RNA产率低、质量差,无法应用于实验操作.本文针对甜叶菊组织次生代谢物多的特点,以传统的CTAB法(cetyl trimethyl ammonium bromide)为基础,分别采用不同的方法进行RNA抽提和纯化.结果发现采用CTAB-LiCl法提取的RNA完整性好,且纯度高;传统的CTAB法提取的RNA完整性好,但有DNA和其它杂质的污染;高盐溶液法提取的RNA降解比较严重,同时还有杂质污染.结论说明CTAB-LiCl法适用于多糖类植物RNA的分离.     

一种简捷提取植物总RNA的准备和操作方法

提取RNA是分子生物学实验中的常用技术。而RNA酶抑制程度成为提取RNA的主要决定因素。准备和操作不当造成的RNA酶污染是实验失败的重要原因。介绍一种简捷的“烧、烤”提取植物总RNA的准备和操作方法,可以有效地抑制外源RNA酶的污染,提取的RNA成功率很高。     

从富含淀粉的水稻胚乳组织中提取功能总RNA 和克隆wee1基因片段的方法研究

水稻胚乳中含有大量的淀粉等物质,用传统的方法提取总RNA难度很大．改良了一种SDS／苯酚法．可以从水稻胚乳中提取高质量的总RNA,解决了RNA易降解、易被污染以及由于总RNA与淀粉等物质共沉淀所造成的低产量等问题．通过分光光度计测量OD值以及变性胶电泳,可以检测出所提取的总RNA质量较高,从1.5g水稻胚乳中可提取到800μg左右的总RNA．提取的总RNA已成功用于RT-PCR克隆目的基因．     

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LiCl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LiCl和乙醇沉淀RNA的效果分析表明,该方法可在10 min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。     

芍药花瓣总RNA的提取

用TRIzol法和改良的热硼酸盐法从芍药幼嫩花瓣中提取总RNA,总RNA的D260nm／D280nm值分别为1．803、1．974。琼脂糖电泳表明,用TRIzol法提取的RNA只有28S、18S2条带,带的亮度差,说明RNA有些降解;而用改良的热硼酸盐法提取的总RNA有28S、18S、5S3条带,带的亮度强,且28S是18S宽度的2倍左右,说明提取的RNA完整、未降解,可用于后续实验。改良的热硼酸盐法更适于从芍药花瓣中提取总RNA。     

The roles of RNA polymerase and RNAase I in stable RNA degradation in Escherichia coli carrying the srnB+ gene

In Escherichia coli cells carrying the srnB+ gene of the F plasmid, rifampin, added at 42 degrees C, induces the extensive rapid degradation of the usually stable cellular RNA (Ohnishi, Y., (1975) Science 187, 257-258; Ohnishi, Y., Iguma, H., Ono, T., Nagaishi, H. and Clark, A.J. (1977) J. Bacteriol. 132, 784-789). We have studied further the necessity for rifampin and for high temperature in this degradation. Streptolydigin, another inhibitor of RNA polymerase, did not induce the RNA degradation. Moreover, the stable RNA of some strains in which RNA polymerase is temperature-sensitive did not degrade at the restrictive temperature in the absence of rifampin. These data suggest that rifampin has an essential role in the RNA degradation, possibly by the modification of RNA polymerase function. A protein (Mr 12 000) newly synthesized at 42 degrees C in the presence of rifampin appeared to be the product of the srnB+ gene that promoted the RNA degradation. In a mutant deficient in RNAase I, the extent of the RNA degradation induced by rifampin was greatly reduced. RNAase activity of cell-free crude extract from the RNA-degraded cells was temperature-dependent. The RNAase was purified as RNAase I in DEAE-cellulose column chromatography and Sephadex G-100 gel filtration. Both in vivo and with purified RNAase I, a shift of the incubation mixture from 42 to 30 degrees C, or the addition of Mg2+ ions, stopped the RNA degradation. Thus, an effect on RNA polymerase seems to initiate the expression of the srnB+ gene and the activation of RNAase I, which is then responsible for the RNA degradation of E. coli cells carrying the srnB+ gene.     

Reconstitution of RNAase P activity using inactive subunits from E. coli and HeLa cells

HeLa cell RNAase P activity found in the flow-through of anti-Sm affinity columns can be separated into inactive RNA and protein components. These components can be used to reconstitute active hybrid enzyme complexes with purified subunits from E. coli RNAase P. The RNA in the HeLa cell fractions employed is enriched for species between 85 and 115 nucleotides long. This reconstitution assay is a convenient means of purifying the functional RNA and protein of HeLa cell RNAase P. Probes derived from the genes for the subunits of E. coli RNAase P hybridize to genomic DNA of gram-negative prokaryotic organisms, but no positive signals are seen with genomic DNA from a variety of eukaryotic organisms.     

The roles of RNA polymerase and RNAase I in stable RNA degradation in Escherichia coli carrying the srnB+ gene

In Escherichia coli cells carrying the srnB⁺ gene of the F plasmid, rifampin, added at 42°C, induces the extensive rapid degradation of the usually stable cellular RNA (Ohnishi, Y., (1975) Science 187, 257–258; Ohnishi, Y., Iguma, H., Ono, T., Nagaishi, H. and Clark, A.J. (1977) J. Bacteriol. 132, 784–789). We have studied further the necessity for rifampin and for high temperature in this degradation. Streptolidigin, another inhibitor of RNA polymerase, did not induce the RNA degradation. Moreover, the stable RNA of some strains in which RNA polymerase is temperature-sensitive did not degrade at the restrictive temperature in the absence of rifampin. These data suggest that rifampin has an essential role in the RNA degradation, possibly by the modification of RNA polymerase function. A protein (M_r 12 000) newly synthesized at 42°C in the presence of rifampin appeared to be the product of the srnB⁺ gene that promoted the RNA degradation. In a mutant deficient in RNAase I, the extent of the RNA degradation induced by rifampin was greatly reduced. RNAase activity of cell-free crude extract from the RNA-degraded cells was temperature-dependent. The RNAase was purified as RNAase I in DEAE-cellulose column chromatography and Sephadex G-100 gel filtration. Both in vivo and with purified RNAase I, a shift of the incubation mixture from 42 to 30°C, or the addition of Mg²⁺ ions, stopped the RNA degradation. Thus, an effect on RNA polymerase seems to initiate the expression of the srnB⁺ gene and the activation of RNAase I, which is then responsible for the RNA degradation of E. coli cells carrying the srnB⁺ gene.     

A rapid and sensitive assay for protein disulphide isomerase activity

An assay procedure for the determination of protein disulphide isomerase activity is presented. The method is based on the reactivation of randomly cross-linked RNAase, the extent of RNAase reactivation being determined from the degradation of radioactively labelled RNA. The method is rapid and sensitive and allows one to test a large number of samples simultaneously.     

How much is secondary structure responsible for resistance of double-stranded RNA to pancreatic ribonuclease A?

1. Double-stranded f2 sus11 or Qbeta RNAs, resistant to bovine pancreatic RNAase A in 0.15 M NaCl/0.015 M sodium citrate (SSC), are quickly and completely degraded at 10-fold lower ionic strength (0.1 X SSC) under otherwise similar conditions. At this ionic strength the secondary structure of double-stranded RNA is maintained, as judged by the following: (a) the unchanged resistance of double-stranded RNA and DNA, under similar low ionic strength conditions, to nuclease S1 from Aspergillus oryzae, in contrast with the sensitivity of the corresponding denatured nucleic acids to this enzyme, specific for single-stranded RNA and DNA; (b) the co-operative pattern of the thermal-transition profile of double-stranded RNA (with a Tm of 89 degrees C) in 0.1 X SSC. 2. Whereas in SSC bovine seminal RNAase (RNAase BS-1) and whale pancreatic RNAase show an activity on double-stranded RNA significantly higher than that of RNAase A, in 0.1 X SSC the activity of the latter enzyme on this substrate becomes distinctly higher than that of RNAase BS-1, and similar to that of whale RNAase. 3. From these results it is deduced that the secondary structure is probably not the only nor the most important variable in determining the susceptibility double-stranded RNA to ribonuclease. Other factors, such as the effect of ionic strength on the enzyme and/or the binding of enzyme to nucleic acids, may play an important role in the process of double-stranded RNA degradation by ribonucleases specific for single-stranded RNA.     

Isolation of intact high-molecular-weight DNA by using guanidine isothiocyanate.

A method for obtaining high-molecular-weight chromosomal DNA from Bacteroides intermedius and Bacteroides gingivalis is described. This technique is a modification of the guanidine isothiocyanate isolation procedure for RNA and should be useful for isolating intact DNA from organisms with high endogenous nuclease activity.     

Isolation of intact high-molecular-weight DNA by using guanidine isothiocyanate

A method for obtaining high-molecular-weight chromosomal DNA from Bacteroides intermedius and Bacteroides gingivalis is described. This technique is a modification of the guanidine isothiocyanate isolation procedure for RNA and should be useful for isolating intact DNA from organisms with high endogenous nuclease activity.     

The putative 15 S precursor of globin mRNA contains a poly (A) sequence

[3H] Uridine or [3H] adenosine pulse-labelled nuclear RNA was isolated from chicken immature red blood cells and separated on denaturing formamide sucrose gradients. RNA of each gradient fraction was hybridized with unlabelled globin DNA complementary to mRNA (cDNA) and subsequently digested by RNAase A and RNAase T1. The experiments revealed two RNA species with globin coding sequences sedimenting 9 S and approx. 15 S, the latter probably representing a precursor of 9 S globin mRNA. A poly (A) sequence was demonstrated in this RNA by two different approaches. Nuclear RNA pulse-labelled with [3H] uridine was fractionated by chromatography on poly (U)-Sepharose. Part of the 15 S precursor was found in the poly(A)-containing RNA. In the second approach 15 S RNA pulse-labelled with [3H]adenosine was hybridized with globin cDNA, incubated with RNAase A and RNAase T1 and subjected to chromatography on hydroxyapatite. The hybrids were isolated and after separation of the strands degraded with DNAase I, RNAase A and RNAase T1. By this procedure poly(A) sequences of approximately 100 nucleotides could be isolated from the 15 S RNA with globin coding sequences. The poly(A) sequence was completely degraded by RNAase T2.