Detection of mutations in human type I collagen mRNA in osteogenesis imperfecta by indirect RNase protection

A method for detecting a wide variety of mutations within type I collagen has been developed and evaluated on a series of patients with osteogenesis imperfecta. RNA, extracted from the nuclear and cytoplasmic compartment of cultured fibroblasts from affected individuals, is hybridized with antisense single-stranded cDNA to the alpha 1(I) mRNA. The hybrid is digested with RNase A and T1 under varying degrees of stringency. The resistant RNA bands are separated by electrophoresis in agarose, transferred to nitrocellulose, and hybridized with antisense cRNA colinear with the protecting probe. This approach is capable of detecting previously defined mutations such as 252-base pair deletion and a 1-base pair mismatch within the alpha 1(I) mRNA. The method appears to be particularly useful in detecting abnormalities of RNA processing that behave as an insert or deletion within the mature mRNA. The procedure should be generally applicable for the identification and localization of any mutation within an entire gene if the gene is expressed as an RNA and a complete cDNA for the mRNA is available.     

Physical and chemical characterization of purified ovalbumin messenger RNA.

Preparative agarose gel electrophoresis under denaturing conditions has been successfully employed to purify large quantities of ovalbumin mRNA from hen oviducts. The mRNA thus prepared is physically homogeneous based on its migration as a single component on electrophoresis in both analytical acid-urea agarose gels and formamide-containing, neutral polyacrylaminde gels; it also sediments as a single peak in sucrose gradients containing 70% formamide. The mRNA is chemically free of ribosomal RNA contamination since its oligonucleotide fingerprint map after complete T1 ribonuclease digestion contains no detectable specific large oligonucleotide markers of ribosomal RNAs. It is also not contaminated by other biologically active messenger RNAs because, when it is added to the cell-free wheat germ translation system, the only protein product synthesized is ovalbumin as analyzed by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate and specific immunoprecipitation. Ovalbumin mRNA has a nucleotide composition of 32.3% A, 21.0% G, 25.7% U, and 20.7% C [(A+U)/(G+C) equal 1.41]. The mRNA contains a heterogeneous poly(A) tract ranging from 20 to 140 residues with a number average chain length of 62 adenylate residues. The molecular weight of the sodium salt of the purified mRNA is approximately 650,000 +/- 63,000, corresponding to a chain length of 1890 +/- 180 nucleotides, as determined by electron microscopy under completely denaturing conditions. This value is in close agreement with the values obtained from: (a) sucrose gradient centrifugation in the presence of 70% formamide; (b) evaluation of poly(A) content in the mRNA and the number average chain length of its poly(A) tract; and (c) sedimentation velocity studies in the presence of 3% formaldehyde. When 125I-labeled ovalbumin mRNA is allowed to hybridize with a large excess of chick DNA, the observed kinetics of hybridization reveal no appreciable reaction between the mRNA and the repeated sequences of the chick DNA, although the mRNA appears to be approximately 600 nucleotides longer than necessary to code for ovalbumin. It thus appears that the entire ovalbumin mRNA is primarily transcribed from a unique sequence in the chick genome.     

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.     

Hybrid selection of mRNA with biotinylated DNA

A novel, convenient, and highly efficient hybrid selection procedure is described. The method utilizes the polymerase chain reaction (PCR) in which one of two primers is biotinylated at the 5'-terminus. The concentration of the biotinylated primer is 100 times that of the other to synthesize biotinylated single-stranded DNA (asymmetric PCR). After hybridization of the biotinylated DNA with mRNA in solution, streptavidin agarose is used to trap the hybrid duplex of mRNA.DNA-biotin onto the solid matrix. The selected mRNA is then eluted from the streptavidin agarose. The quantitative physical recovery of selected mRNA is about 70% with about 33% retention of biological activity.     

一种改进的异硫氰酸胍法结合硅胶膜离心吸附柱提取孕妇外周血微量胎儿RNA的方法

孕妇外周血中存在胎儿RNA为无创性产前诊断提供了基础.但血液中富含RNA酶和微量胎儿RNA的特点,对从孕妇血浆中提取胎儿RNA带来困难. 我们以ε血红蛋白基因和胎盘特异表达基因4（PLAC4）mRNA作为研究对象,用改进的异硫氰酸胍法结合硅胶膜离心吸附柱法探索孕妇外周血中胎儿微量RNA的提取方法,获得满意效果. 30例孕妇和9例非孕妇外周血样品中总RNA经凝胶电泳测定显示3条带,分别为28S, 18S和 5.8S. 其28S条带亮度为18S亮度的2倍.总RNA质量浓度（A260／A280）为1.97 g/L,光密度比值(A260-A320）/（A280- A 320）为1.86. 30例孕妇外周血样本有7例提取到ε血红蛋白基因mRNA,ε血红蛋白基因 mRNA 的最小浓度为0.537 μg/mL,最大浓度为1.79 μg/mL,ε血红蛋白基因mRNA的浓度中位数为124 μg/mL. 30例孕妇外周血样本提取到PLAC4 基因mRNA,浓度最小值为2.105×103 copies/mL,最大值为12.760×103 copies/mL,而9例非孕妇中均未提取到（P＜0.01）,浓度中位数为6.612×103 copies/mL. 因此,改进的异硫氰酸胍法与硅胶膜离心吸附柱纯化法相结合,可有效抑制RNA降解,用于提取、纯化孕妇血液中微量胎儿RNA.     

用低浓度硫氰酸胍提取高质量植物RNA

目前分离RNA的方法很多, 但大多是基于动物材料建立的方法, 而针对植物材料的方法并不多见. 建立了一种从植物材料中分离高质量RNA的硫氰酸胍/氯化锂/热酚法. 与其他硫氰酸胍的方法相比, 该方法所用硫氰酸胍的浓度仅为现有方法的1/40 (0.1 mol/L),降低了实验成本, 且所得RNA质量令人满意. 用该方法分离的RNA在琼脂糖凝胶电泳上可清晰分出4条核糖体RNA (rRNA)带; 用此RNA进行RNA印迹或分离mRNA进行体外翻译试验, 均获得很好效果.     

仿刺参体壁总RNA提取方法的建立

目的:通过对TRIzol一步法进行改进,建立一种从富含胶原蛋白、多糖及色素的仿刺参体壁提取总RNA的有效方法。方法:样品在液氮中研磨并用TRIzol匀浆后再进行抽提;对TRIzol一步法提取的总RNA进行DNaseⅠ消化和酚氯仿抽提,用2.5mol/L的醋酸钾沉淀,并加入适量糖原(10mg/mL)与RNA共沉淀。结果:琼脂糖凝胶电泳和紫外分光光度法以及RT-PCR检测结果表明,改进的方法能够有效去除基因组DNA、蛋白、多糖及色素的污染,RNA的产率提高。结论:制备的总RNA纯度高,完整性好,能够满足mRNA差异显示RT-PCR等分子生物学研究的要求,是一种提取仿刺参体壁及其他富含黏多糖、胶原蛋白和色素的动物组织总RNA的有效方法。     

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 new blotting medium for the simple isolation and identification of highly resolved messenger RNA.

A simple method has been developed that allows the rapid isolation and identification of highly resolved mRNA molecules. RNA species are separated by gel electrophoresis and then blotted on to a paper sheet to which polyuridylic acid has been covalently bound. This mRNA affinity paper ("mAP") specifically binds, in a reversible manner, polyA+ containing molecules. A replica picture of the agarose gel is thus obtained on the mAP, from which bound mRNA molecules can be eluted by heating in water. In addition to their simple isolation individual mRNA species, whilst still bound to mAP, can be identified by both "in-situ" hybridization and translation.     

Northern blot mapping: a procedure for mapping mRNA immobilized on nitrocellulose by probing with end-labeled DNA fragments

A simple method for mapping RNA on a Northern blot with a mixture of end-labeled DNA fragments is described. The DNA fragments are labeled either in 5' or in 3' directly after digestion by restriction enzyme(s) and used without any further purification step as probe to hybridize a Northern blot. After autoradiography, the DNA fragments hybridized to each mRNA species are recovered by heating the nitrocellulose and analyzed on denaturing polyacrylamide or agarose gels. This method indicates which DNA fragment hybridizes with which mRNA species and requires far fewer different manipulations than successive hybridization of a Northern blot with several nick-translated purified DNA fragments.     

Measurement of mRNA by quantitative PCR with a nonradioactive label.

This report describes the development of a method to measure mRNA in small samples of human tissue by the polymerase chain reaction with a nonradioactive label. In this method RNA is reverse-transcribed in the presence of a control RNA, and subsequently amplified by the polymerase chain reaction during which a nonradioactive label (digoxigenin-11-dUTP) is incorporated. Gel blotting and immunological detection of digoxigenin followed by a chemiluminescent reaction provide an intense signal on film. This allows the detection and quantitation of 3-hydroxy-3-methylglutaryl (HMG) CoA reductase mRNA in 12 ng of RNA. We demonstrate that this is a sensitive and reproducible method, and that quantitation is linear with respect to the amount of mRNA present. The application of this method to the measurement of low density lipoprotein receptor and 3-hydroxy-3-methylglutaryl coenzyme A reductase mRNA levels in circulating peripheral blood mononuclear cells and human liver biopsy samples is discussed. The use of chemiluminescent reagents instead of radioactive labels allows this procedure to be performed safely in laboratories not equipped for radioactivity.     

A rapid method to quantitate non-labeled RNA species in bacterial cells

We have developed a rapid method to quantitate specific bacterial RNA species. The method measures the steady-state level of RNA, produces a linear response over more than a 16-fold range of RNA concentration, and can be used for Staphylococcus aureus, Escherichia coli and Bacillus subtilis. In this method, a sheared whole-cell lysate of approx. 7 x 10(8) organisms, prepared as for plasmid screening, is separated on agarose, blotted to a nitrocellulose filter, hybridized with a radiolabeled DNA probe, and autoradiographed. The RNA species are quantitated by counting the radioactive bands on the filter. We have applied the method to the measurement of mRNA induction of the genes encoding beta-lactamase, ermC rRNA methylase, and the alpha-complementing fragment of beta-galactosidase. Upon induction, a ten-fold increase in the mRNA for each gene was observed. The peak mRNA level occurred after 30 min for beta-lactamase, 20 min for beta-galactosidase, and 5 min for the ermC rRNA methylase.     

北京鸭卵对蛋白mRNA的提纯及某些性质的鉴定

 本文报道了从产卵期北京鸭输卵管中提取总RNA,经Olilo(dT)-纤维素柱层析,再经sepharose 4B柱层析步骤,得到了纯化的鸭卵清蛋白mRNA。我们建立了麦胚无细胞体系并探索了鸭卵清蛋白mRNA在此体系中翻译的最适条件。在此条件下测定了各纯化步骤的总mRNA翻译活性,并用免疫沉淀法测定其中卵清蛋白mRNA的活性。测定结果表明我们从mRNA中分离得到了纯鸭卵清蛋白mRNA。用变性的琼脂糖凝胶电泳对各纯化步骤的核酸样品进行组成分析,确定出鸭卵清蛋白mRNA的大小约为21S。