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.     

Enhanced sensitivity RNA gel loading buffer that enables efficient RNA separation on native gels

RNA gel analysis is essential for quality assessment of RNA preparations for subsequent analysis such as microarrays and real-time PCRs. The routinely used standard electrophoresis of RNA through formaldehyde-containing agarose gels is not only labor-intensive and time-consuming, but also involves sizeable quantities of hazardous materials. Above all, it is not sensitive, requiring more than 1 microgram of RNA for the assay. Current gene expression profiling with microarrays and real-time PCR often involves limiting amounts of RNA. It is therefore important to have a more sensitive way to analyze RNA. Here we report an improved ethidium bromide-based RNA gel analysis system with our Superload buffer that increases sensitivity to 12.5 ng of total RNA and allows RNA analysis on a regular native Tris-acetate EDTA (TAE) agarose gel.     

Separation of long RNA by agarose–formaldehyde gel electrophoresis

We describe a method to facilitate electrophoretic separation of high-molecular-weight RNA species, such as ribosomal RNAs and their precursors, on agarose–formaldehyde gels. Two alternative “pK-matched” buffer systems were substituted for the traditionally used Mops-based conductive medium. The key advantages include shortened run times, a 5-fold reduction in formaldehyde concentration, a significantly improved resolution of long RNAs, and consistency in separation. The new procedure has a streamlined work flow that helps to minimize errors and is broadly applicable to agarose gel electrophoresis of RNA samples and their subsequent analysis by Northern blotting.     

Agarose gel electrophoresis of denatured RNA with silver staining

This paper describes agarose gel electrophoresis and silver staining of denatured RNAs. Glyoxal- or formaldehyde-denatured RNAs are electrophoresed in an agarose gel cast on a plastic support using an inert, low conductivity buffer. Following electrophoresis, the gel is stained with a sensitive silver stain. The method produces sharp, well-resolved bands and yields accurate RNA size estimates. Because of its sensitivity and simplicity, it is suitable for routine laboratory use.     

Denaturing RNA electrophoresis in TAE agarose gels

Current methods of analytical RNA electrophoresis are based on the utilization of either complicated laboratory instrumentation or toxic, carcinogenic, or expensive chemicals. We suggest here the use of classical Tris-acetate-ethylenediamine tetraacetic acid (TAE) agarose gels combined with prior denaturation of RNA samples in hot formamide for the electrophoretic separation of RNA species. We present a brief comparison of the proposed TAE/formamide method with the most common 3-(N-morpholino)propanesulfonic acid/formaldehyde agarose gel protocol and show that both methods produce comparable results for size determination of RNA molecules and subsequent Northern blotting of gels. In addition to purified RNA samples, the robustness of the TAE/formamide protocol is demonstrated by its suitability for the analysis of RNA quality in crude yeast cell lysates containing large amounts of proteins, DNA, and other contaminating molecules. We therefore propose the TAE/formamide agarose electrophoresis as a rapid, simple, and cheaper alternative to current methods of RNA electrophoresis. Additionally, another benefit is the reduced exposure of laboratory personnel to hazardous chemicals.     

Two-dimensional gel electrophoresis to separate large RNA molecules such as messenger RNAs

This paper describes a two-dimensional gel electrophoresis method for separating large RNA molecules such as messenger RNAs. RNAs were at first separated on a polyacrylamide plus agarose composite gel and subjected to a second dimension electrophoresis on a polyacrylamide gel containing urea. This method is illustrated by analyses of poly(A)+ yeast RNAs. About 80 discrete spots were detected on the gel, when RNAs from 1000 to 3500 nucleotides in size were examined.     

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 .     

荧光Real-time PCR鉴定鸡胸骨总RNA质量

为了更准确地鉴定提取鸡骨总RNA的质量,试验分别用核酸蛋白检测仪、1%琼脂糖凝胶电泳和荧光Real-time PCR检测评价3种不同的方法提取成年鸡胸骨总RNA的质量。结果显示,荧光Real-time PCR可更好地鉴定提取的骨总RNA的质量。用核酸蛋白检测仪、1%琼脂糖凝胶电泳能粗略检测RNA的纯度和完整性,但不能反映提取过程中是否引起基因不均一的减少,所检测的纯度也不能精确反映RNA的反转录效率。     

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.     

SELECTIVE RETENTION AND FILTRATION OF BRAIN NUCLEIC ACIDS IN AGAROSE GELS

Abstract— Total nucleic acids of rat brain have been separated by agarose gel chromatography at 2 m -NaCl into DNA. transfer RNA plus low molecular weight RNA. and high molecular weight RNA fractions. The DNA fraction contained less than 1 per cent RNA by weight judged by either short-term or long-term labelling with ortho[³²P]phosphate. The high molecular weight RNA fraction contained 28 s and 18 s ribosomal RNAs and a heterogeneous population of 20-60 s RNAs, apparent after short-term labelling and characterized by a high content of nearest-neighbour-labelled uridylic acid. The rapidly sedimenting (>30 s ) portion of these RNAs could be largely separated from ribosomal RNAs by gel filtration using 4% agarose. The ribosomal RNAs could be fully resolved into 28 s and 18 s components by agarose gel chromatography at 0.5 m -0.6 m -NaCl, as shown by analysis of their sedimentation and nucleotide composition.     

RNA甲醛琼脂糖凝胶电泳的优化及探讨

目的:优化RNA甲醛琼脂糖凝胶电泳条件。方法:对实验器材进行彻底的RNase灭活处理;在原有电泳过程的基础上,增加预电泳、缓冲液液面的处理和制胶过程中未加入溴化乙锭等优化过程。结果:优化后的电泳不仅能验证总RNA的完整性,而且能鉴定RNA的分子大小。结论:优化的RNA甲醛琼脂糖凝胶电泳法操作简单、省时、快速,RNA条带清晰、定位准确、无弥散及非特异条带。     

RNA甲醛琼脂糖凝胶电泳的优化及探讨

目的：优化RNA甲醛琼脂糖凝胶电泳条件。方法：对实验器材进行彻底的RNase灭活处理;在原有电泳过程的基础上,增加预电泳、缓冲液液面的处理和制胶过程中未加入溴化乙锭等优化过程。结果：优化后的电泳不仅能验证总RNA的完整性,而且能鉴定RNA的分子大小。结论：优化的RNA甲醛琼脂糖凝胶电泳法操作简单、省时、快速,RNA条带清晰、定位准确、无弥散及非特异条带。     

RNA在离体小鼠肝脏组织中的稳定性

采取肝脏等临床样本时,常因不能及时保存造成核酸降解,从而影响后续实验的进行.本研究旨在通过对室温条件下放置不同时间的小鼠肝脏组织的RNA的完整性进行评价,为肝脏临床样本的采集与保存提供依据.将离体小鼠肝脏组织在室温下放置0～180min后,提取总RNA,采用电泳、RT-PCR和芯片生物分析仪检测RNA的完整性.电泳结果显示,将小鼠肝脏置于室温180min后,RNA尚未发生降解.对β肌动蛋白,GAPDH和Trp53的RT-PCR的分析表明,室温下保存180min的RNA样品均未降解.生物分析仪检测的RNA完整性系数(RIN)都大于7.9,样品可用于后续研究.因此,离体后的小鼠肝脏置于室温下3h以内,RNA仍能保持其完整性.     

Detection of proteases by clotting of casein after gel electrophoresis

Clotting of casein provides a sensitive method for detection of proteases after gel electrophoresis. The method is here designated "caseogram." After electrophoresis the gel was equilibrated with 0.15-0.3 M sodium acetate, pH 5.3, and an 1% agarose gel containing 1% skim-milk powder in 0.1 M sodium acetate, pH 5.3, was placed on top of the electrophoresis gel. By incubation at 37 degrees C for 2 h the protease-containing zones produced distinct precipitates in the skim-milk gel. For permanent documentation the skim-milk gel was stained with amido black. The detection limit for pepsin A is 5 ng in the caseogram against 25 ng by hemoglobin digestion at pH 2.5. For calf chymosin it is 1 ng against 100 ng by digestion of hemoglobin at pH 3.5. Caseograms work well after agar gel electrophoresis, after different types of immunoelectrophoresis, and after isoelectric focusing or disc electrophoresis in polyacrylamide gels. Since inert proteins do not interfere with the detection, the method is especially suitable for analysis of crude samples. Samples containing pepsinogen or pepsinogen-like zymogens may be activated at pH 2 before equilibration at pH 5.3.     

RNA synthesis in maturing avian erythrocyte nuclei

The rate of RNA synthesis and its inhibition by α-amanitin in the nuclei of mature and immature avian erythrocytes are increased with the increase in ionic strength of incubation medium. Polyacrylamide gel electrophoresis indicates that heterogeneous species of RNAs are synthesised in the mature and immature erythrocyte nuclei. However, a large number of high molecular weight RNAs are synthesised in the nuclei of immature erythrocytes. Elution profiles on poly(U)-sepharose chromatography indicate that the RNAs synthesised in the nuclei of two types of cell contain poly(A) segments. Sixteen per cent of mature erythrocyte nuclear RNA syntbesised are polyadenylated, while it is 13% in immature erythrocyte nuclei. However, the total RNA synthesised is 2–3 fold higher in immature erythrocyte nuclei than that in mature erythrocyte nuclei.     

Cell-free translation of murine coronavirus RNA.

The coding assignments of the intracellular murine hepatitis virus-specific subgenomic RNA species and murine hepatitis virion RNA have been investigated by cell-free translation. The six murine hepatitis virus-specific subgenomic RNAs were partially purified by agarose gel electrophoresis and translated in an mRNA-dependent rabbit reticulocyte lysate, and the cell-free translation products were characterized by gel electrophoresis, immunoprecipitation, and tryptic peptide mapping. These studies have shown that RNA 7 codes for the nucleocapsid protein, RNA 6 codes for the E1 protein, RNA 3 codes for the E2 protein, and RNA 2 codes for a 35,000-dalton nonstructural protein. Genomic RNA directs the cell-free synthesis of three structurally related polypeptides of greater than 200,000 in molecular weight.     

Improved non-radioactive Northern blot protocol for detecting low abundance mRNAs from mammalian tissues

A rapid and sensitive non-radioactive Northern blot protocol is described which has been optimised in several critical steps. This is based on a formaldehyde-denaturing agarose gel electrophoresis, an alkaline transfer, hybridisation with digoxigenin-DNA probes and detection with a chemiluminescent substrate. This method allows even low abundance mRNAs to be detected in total RNA samples from mammalian tissues.     

Modification of Turnip yellow mosaic virus coat protein and its effect on virion assembly

Turnip yellow mosaic virus (TYMV) is a positive strand RNA virus. We have modified TYMV coat protein (CP) by inserting a c-Myc epitope peptide at the N- or C-terminus of the CP, and have examined its effect on assembly. We introduced the recombinant CP constructs into Nicotiana benthamiana leaves by agroinfiltration. Examination of the leaf extracts by agarose gel electrophoresis and Western blot analysis showed that the CP modified at the N-terminus produced a band co-migrating with wild-type virions. With C-terminal modification, however, the detected bands moved faster than the wild-type virions. To further examine the effect, TYMV constructs producing the modified CPs were prepared. With N-terminal modification, viral RNAs were protected from RNase A. In contrast, the viral RNAs were not protected with C-terminal modification. Overall, the results suggest that virion assembly and RNA packaging occur properly when the N-terminus of CP is modified, but not when the C-terminus is modified. [BMB Reports 2013; 46(10): 495-500]     

GelStar nucleic acid gel stain: high sensitivity detection in gels.

GelStar nucleic acid gel stain can be used for sensitive fluorescent detection of both double-stranded (ds) and single-stranded (ss) DNAs, oligonucleotides and RNA in gels. The stain can be added to agarose gels at casting for immediate imaging after electrophoresis or can be used after electrophoresis with both agarose and acrylamide gels. GelStar stain is highly fluorescent only when bound to nucleic acids thus giving superior signal-to-noise ratios and obviating the need to destain the gel. The detection limits of GelStar strain are 20 pg for dsDNA, 25 pg for ssDNA and 10 ng for native or glyoxal-treated RNA.     

Dimerization of human immunodeficiency virus type 1 RNA involves sequences located upstream of the splice donor site.

The retroviral genome consists of two homologous RNA molecules associated close to their 5' ends. We studied the spontaneous dimerization of four HIV-1 RNA fragments (RNAs 1-707, 1-615, 311-612, and 311-415) containing the previously defined dimerization domain, and a RNA fragment (RNA 1-311) corresponding to the upstream sequences. Significant dimerization of all RNAs is observed on agarose gels when magnesium is included in the electrophoresis buffer. In contrast to dimerization of RNAs 311-612 and 311-415, dimerization of RNAs 1-707, 1-615 and 1-311 strongly depends on the size of the monovalent cation present in the incubation buffer. Also, dimerization of RNAs 1-707, 1-615, and 1-311 is 10 times faster than that of RNAs 311-612 and 311-415. The dimers formed by the latter RNAs are substantially more stable than that of RNA 1-615, while RNA 1-311 dimer is 5-7 degrees C less stable than RNA 1-615 dimer. These results indicate that dimerization of HIV-1 genomic RNA involves elements located upstream of the splice donor site (position 305), i.e. outside of the previously defined dimerization domain.