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.     

Comparative analyses of Saccharomyces cerevisiae RNAs using Agilent RNA 6000 Nano Assay and agarose gel electrophoresis

Precise quantification and quality characterisation of isolated RNAs are prerequisites for their further exploitation in genome-wide microarrays, Northern blots, cDNA library preparation and others. Our data indicate that RNA analyses using Agilent RNA Nano Assay exhibit several advantages when compared with those performed on ethidium bromide-stained agarose gel electrophoresis or on a spectrophotometer. The RNA Nano Assay makes it possible to estimate RNA concentrations in the range from 1000 ng microl(-1) to 17 ng microl(-1). The presence of impurities including traces of DNA within RNA samples does not influence the concentration measurements. Like agarose gel electrophoresis, RNA Nano Assay allows to analyse RNAs dissolved in formamide and therefore protected against RNase action. Moreover, it allows a clearer distinction of partially degraded samples. The limitation of RNA Nano Assay is the impossibility to detect and to analyse double-stranded RNAs.     

大鼠及小鼠微卫星引物在社鼠中的跨种扩增

利用微卫星引物在同一属、科、目不同种之间具有通用性的特点,通过PCR扩增、聚丙烯凝胶电泳和银染技术对社鼠(Niviventer confucianus)近缘物种大鼠(Rattus norvegicus)、小鼠(Mus musculus)中已知的70个微卫星位点引物进行跨种扩增,筛选适合社鼠相关研究的多态微卫星引物.结果发现,40个位点引物出现扩增条带,21个位点引物能够稳定扩增,其中15个位点杂合,13个位点具有多态性;PCR扩增的Mg2+浓度主要集中在1.5及2.0 mmol/L,退火温度在50～60℃之间不等.虽然部分扩增产物有影子带的存在,但并不影响等位基因的判读.总体来看,利用大、小鼠的微卫星引物扩增社鼠的微卫星位点是可行的.     

Enhanced resolution of random amplified polymorphic DNA genotyping of Pseudomonas aeruginosa

AIMS: To develop a rapid, sensitive and reproducible screening test for the detection of nosocomial spreading of Pseudomonas aeruginosa. METHODS AND RESULTS: Ps. aeruginosa genomic DNA extraction, RAPD-PCR, electrophoresis on acrylamide gel and silver staining were performed by using standardized reagents and conditions. The results were compared with the agarose gel electrophoresis followed by ethidium bromide staining. CONCLUSIONS: The coupling of acrylamide gel electrophoresis and silver staining gave about 80% more DNA bands than the traditional method, allowing a finer discrimination among different Ps. aeruginosa strains. SIGNIFICANCE AND IMPACT OF THE STUDY: By enhancing the resolution of the electrophoretic separation and the sensitivity of the staining, random amplification could be easily applied to the surveillance and prevention of nosocomial infections by clinical microbiology laboratories.     

检测植物DNA扩增多态性方法的比较和改进

以辽东栎(Quercus liaotungensis Koidz.)、锦鸡儿(Cargagana ssp.)和野大豆(Glycine soja(L.)Sieb.etZucc.)为材料,比较了随机扩增多态DNA(RAPD)和DNA扩增指纹(DAF)方法。用RAPD的琼脂糖胶电泳和溴乙锭染色,RAPD和DAF谱一般不足10条带。用DAF的变性聚丙烯酰胺凝胶电泳(PAGE)和银染,极大地提高了RAPD的灵敏度和分辨率,多达20～40个产物。用3＇末端完全相同的引物,RAPD和DAF有同样的扩增谱,说明两种方法有相似的机理。降低胶的浓度可提高RAPD和DAF的分辨率,达40～80条带。琼脂糖电泳分离的溴乙锭显示的单荧光带,用PAGE和银染可分辨出多个片段。分子克隆证实单荧光带的分子量异质性。在用Taq DNA多聚酶的条件下,RAPD和DAF的再现性均良好。     

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.     

An electrophoretic karyotype and assignment of ribosomal genes to resolved chromosomes of Pneumocystis carinii

Pulsed-field gel electrophoresis was used to generate a molecular karyotype of chromosomes from the opportunistic AIDS pathogen, Pneumocystis carinii. P. carinii cysts and trophozoites were isolated from immunosuppressed rats, lysed in situ in agarose blocks, and subjected to orthogonal-field gel electrophoresis (OFAGE) and contour-clamped homogeneous-field gel electrophoresis (CHEF). OFAGE and CHEF gels resolved, respectively, 16 or 20 chromosome bands ranging in size from 0.32-1.5 megabase pairs. Summation of the estimated sizes of these chromosomes suggested a total genome complexity for P. carinii of 8-16 megabase pairs. Homologous probes for the genes encoding the 18S, 5.8S, and 5S ribosomal RNAs were hybridized to filter blots of the pulsed-field gels to map these genes to specific P. carinii chromosomes.     

Analysis of the electrophoretic properties of double-stranded DNA and RNA in agarose gels at a finite voltage gradient

The electrophoretic mobilities of double-stranded (ds) DNAs and ds RNAs of various lenths, L, were measured in gels of 0.4–1.8% (w/v) agarose at a voltage gradient of 1.0 V/cm. Differences in the electrophoresis of ds DNA and ds RNA are presented and discussed. A general expression is derived that describes the electrophoretic mobility, M, of either type of ds nucleic acid as a function of the gel concentration and the nucleic acid length: M = M′₁(L/L₀)^?x ? M₂, where M′₁ and L₀ are constants, and x and M₂ depend on the agarose gel concentration. The results obtained by fitting our data with this equation are consistent with the mobilities of nucleic acids in a wide range of gel concentrations, including free electrophoresis in solution and electrophoresis in gles of high agarose concentration in which nuleic acids are expected to reptate through the gel matrix. Finally, various methods of plotting agarose gel electrophoresis data are discussed.     

Characterization of messenger RNA by direct translation from agarose gels

A method for characterizing nanogram quantities of poly(A)-containing messenger RNAs that have been fractionated according to size by electrophoresis through agarose gels has been developed. The mRNAs from Friend leukemia cells were identified by the protein products they encode, as determined by slicing the agarose gel and directly translating the enclosed mRNA with an extract from rabbit reticulocytes that had been treated with micrococcal nuclease. A number of parameters which affect the efficiency of translation in this system have been examined. These include the sensitivity of the in vitro translational system to RNA, the agarose concentration, the incubation temperature, and the addition of either exogeneous tRNA or RNasin. The procedure is rapid, simple, reproducible, and applicable for the fractionation and characterization of mRNAs from any source.     

Agarose Gel Electrophoresis for the Separation of DNA Fragments

Agarose gel electrophoresis is the most effective way of separating DNA fragments of varying sizes ranging from 100 bp to 25 kb¹. Agarose is isolated from the seaweed genera Gelidium and Gracilaria, and consists of repeated agarobiose (L- and D-galactose) subunits². During gelation, agarose polymers associate non-covalently and form a network of bundles whose pore sizes determine a gel''s molecular sieving properties. The use of agarose gel electrophoresis revolutionized the separation of DNA. Prior to the adoption of agarose gels, DNA was primarily separated using sucrose density gradient centrifugation, which only provided an approximation of size. To separate DNA using agarose gel electrophoresis, the DNA is loaded into pre-cast wells in the gel and a current applied. The phosphate backbone of the DNA (and RNA) molecule is negatively charged, therefore when placed in an electric field, DNA fragments will migrate to the positively charged anode. Because DNA has a uniform mass/charge ratio, DNA molecules are separated by size within an agarose gel in a pattern such that the distance traveled is inversely proportional to the log of its molecular weight³. The leading model for DNA movement through an agarose gel is "biased reptation", whereby the leading edge moves forward and pulls the rest of the molecule along⁴. The rate of migration of a DNA molecule through a gel is determined by the following: 1) size of DNA molecule; 2) agarose concentration; 3) DNA conformation⁵; 4) voltage applied, 5) presence of ethidium bromide, 6) type of agarose and 7) electrophoresis buffer. After separation, the DNA molecules can be visualized under uv light after staining with an appropriate dye. By following this protocol, students should be able to: 1. Understand the mechanism by which DNA fragments are separated within a gel matrix 2. Understand how conformation of the DNA molecule will determine its mobility through a gel matrix 3. Identify an agarose solution of appropriate concentration for their needs 4. Prepare an agarose gel for electrophoresis of DNA samples 5. Set up the gel electrophoresis apparatus and power supply 6. Select an appropriate voltage for the separation of DNA fragments 7. Understand the mechanism by which ethidium bromide allows for the visualization of DNA bands 8. Determine the sizes of separated DNA fragments       

一步法电泳分析肌联蛋白亚型和肌球蛋白重链

目的为研究超大分子量肌小节蛋白肌联蛋白(titin)的生理病理功能,在一次电泳过程中同时分离titin各亚型和中分子量肌小节蛋白肌球蛋白重链(myosin heavy chain,MHC)。方法使用16cm×18cm垂直电泳系统,在电泳板下1/3灌注10g/L SDS-PAGE胶,上2/3灌注60g/L SDS-琼脂糖(SDS-VAGE)胶。低温8℃下持续电泳5h,在电泳板上层以SDS-VAGE胶电泳分离titin亚型,下层以SDS-PAGE胶电泳分离MHC。电泳后VAGE胶使用银染法标记titin各亚型,PAGE胶使用考马斯亮蓝染色法标记MHC。结果 titin各亚型得到有效的分离,目标蛋白条带显示清晰,与其分子量大小一一对应,分离效果明确。结论一步法垂直电泳系统可应用于超大分子量蛋白的电泳,同时可分离多个分子量差距大的蛋白,提高蛋白电泳实验效率。     

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.     

The synthesis of vitellogenins by the digestive gland of Helix aspersa: evidence from cell-free translation of mRNA.

All the RNAs were extracted separately from the gonads and digestive glands of the garden snail Helix aspersa. After separation by oligo-dT-cellulose chromatography, the mRNA were evaluated using optical density measurements and agarose gel electrophoretic analysis and then used in a cell-free translation system: a rabbit reticulocyte lysate added with 35S methionine. The in vitro synthesized proteins were characterized by immunoprecipitation with polyclonal antibodies which were raised against mature oocyte proteins. The proteins were then evaluated quantitatively using liquid scintillation counting and qualitatively using SDS polyacrylamide gel electrophoresis and fluorography. The results demonstrated that the digestive gland of the garden snail is a vitellogenin synthesis site.     

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.     

核酸染料Genefinder使用方法的比较

目的:使用核酸染料Genefinder检测琼脂糖凝胶中的核酸,通过比较预染样品法、胶内染色法和后染法三种染色方法的染色效果,了解该染料的染色特性,以期找到性能稳定,染色效果好的染色方法.方法:在琼脂糖凝胶电泳中,以不同的染色方法使用核酸染料Genefinder进行染色,对染色结果进行比较分析.结果:使用电泳后染色方法染色效果较好,胶内染色法次之,预染样品法效果最差.结论:核酸染料Genefinder会干扰DNA的迁移效率,因此,使用Genefinder进行电泳后染色是一种较好的染色方法.     

Gel electrophoretic fractionation of RNAs by partial denaturation with methylmercuric hydroxide.

The changes in agarose gel electrophoretic velocities of several RNAs of varying molecular weight and base composition with concentration of the denaturant, methylmercuric hydroxide (MMH), have been studied. At intermediate MMH concentrations, the mobility of any one species is intermediate between the “native” (no MMH) and fully denatured (5 mm MMH) values. A + U-richer RNAs are partially denatured at lower concentrations of MMH than are G + C-richer RNAs. Electrophoresis at intermediate MMH concentrations is useful for resolving some RNA species that are not well resolved either in the absence of MMH or under fully denaturing (5 mm MMH) conditions. It is shown that it is possible to carry out MMH electrophoresis in polyacrylamide gels; this is useful for low molecular weight RNAs. An equation to correlate changes in mobility between the native and denatured states is proposed.     

A sensitive silver stain for proteins in agarose gels

A silver stain for proteins in agarose gels which is at least 10 times as sensitive as Coomassie blue is described. The method is simple to use and is particularly useful for the study of protein bands in the gamma region on electrophoresis of fluids such as cerebrospinal fluid in which the protein concentration is low. It readily detects bands of IgG containing 20 to 40 ng/band (approx 3 to 6 ng of IgG/mm2 of gel).