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1.
A rapid, inexpensive and reliable method for total RNA extraction from fruiting bodies of Lentinula edodes containing large quantities of polysaccharides and secondary metabolites is described. An initial extraction step using saturated NaCl solution facilitates the separation of nucleic acids from contaminants and, after further extraction with organic solvents and precipitation with 2-propanol, total RNA of high purity and suitable for applications such as cDNA synthesis, RT-PCR and Northern blot hybridization was obtained. The procedure may also have wider applicability for total RNA extraction from the tissues of other mushrooms. 相似文献
2.
The antisense therapeutic strategy makes the assumption that sequence-specific hybridization of an oligonucleotide to its target can take place in living cells. The present work provides a new method for the detection of intracellular RNA molecules using in situ hybridization on living cells. The first step consisted in designing nonperturbant conditions for cell permeabilization using streptolysin O. In a second step, intracellular hybridization specificity was evaluated by incorporating various types of fluorescently labeled nucleic acid probes (plasmids, oligonucleotides). Due to its high expression level, the 28S ribosomal RNA was retained as a model. Results showed that: (1) no significant cell death was observed after permeabilization; (2) on living cells, 28S RNA specific probes provided bright nucleoli and low cytoplasmic signal; (3) control probes did not lead to significant fluorescent staining; and (4) comparison of signals obtained on living and fixed cells showed a colocalization of observed fluorescence. These results indicate the feasibility of specific hybridization of labeled nucleic acid probes under living conditions, after a simple and efficient permeabilization step. This new detection method is of interest for investigating the dynamics of distribution of various gene products in living cells, under normal or pathological conditions.Abbreviations PI
propidium iodide
- SLO
streptolysin O 相似文献
3.
Samia Daldoul Synda Chenenanoui Ahmed Mliki Michael H?fer 《Acta Physiologiae Plantarum》2009,31(4):871-875
A method is described, which consistently yields high quality total RNA from grapevine. Dissolving of crude RNA pellets in
borate-containing buffer, instead of normally used water before a selective lithium chloride precipitation, was found to be
a critical step, leading to a 2.5-fold increase of yield. The resulting RNA preparations were suitable for standard downstream
applications and also for cDNA library construction. The method worked efficiently and reproducibly and could easily be scaled
from milligram to gram quantities of plant material grown in hydroponic culture, sandy soil or Perlite. It was applied to
different kinds of grapevine tissues (leaves, stem) and, after additional adaptation of the protocol, to roots. 相似文献
4.
Sánchez-Rodríguez A Portal O Rojas LE Ocaña B Mendoza M Acosta M Jiménez E Höfte M 《Molecular biotechnology》2008,40(3):299-305
Efficient RNA isolation is a prerequisite for gene expression studies and it has an increasingly important role in the study
of plant–fungal pathogen interactions. However, RNA isolation is difficult in filamentous fungi. These organisms are notorious
for their rigid cell walls and the presence of high levels of carbohydrates, excreted from the fungal cells during submerged
growth, which interferes with the extraction procedures. Although many commercial kits are already available for RNA isolation,
they do not provide, in most cases, enough amount of pure RNA to be used in upstream applications. In the present work, we
propose an easy and efficient protocol for isolating total RNA from the filamentous fungus Mycosphaerella fijiensis, the most important foliar pathogen of Musa spp. varieties worldwide. In addition, we applied the proposed protocol to the isolation of total RNA from banana leaves
infected with the pathogen. Our methodology was developed based on the SDS method with modifications including a carbohydrate
precipitation step. The protocol resulted in high-quality total RNA, from fungal mycelium grown in PDB medium and infected
banana leaves, suitable for further molecular studies. The proposed methodology is also applicable to the ascomycete fungus
Passalora fulva (syn. Cladosporum fulvum).
Aminael Sánchez-Rodríguez and Orelvis Portal contributed equally to the article. 相似文献
5.
The introduction of modified or labeled nucleotides into RNA is a powerful RNA engineering tool as it enables us to investigate how native RNA modifications affect RNA function and structure. It also helps in the structural analysis of RNA. A modified nucleotide can be introduced into a specific position of RNA by the method of two-step enzymatic ligation of RNA fragments. However, this method requires a complicated purification step between the two ligation steps that results in low yields of the ligation product. Here we have developed a new ligation technique employing periodate oxide that eliminates this purification step. This increases the total yield of the ligation product and makes it a faster procedure. 相似文献
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8.
Interaction between RNA polymerase and a ribosomal RNA promoter of E. coli. 总被引:3,自引:2,他引:1
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The interaction between RNA polymerase and the E. coli ribosomal (r) RNA promoter(s) of the rrnE operon has been studied by the filter-binding method. The extent of complex formation between RNA polymerase and rrnE promoter(s) is salt-dependent; ppGpp specifically inhibits interaction of RNA polymerase with the rrnE promoter(s). A tentative model is proposed for the molecular events in the early steps of rRNA initiation: a transition of the primarily formed, labile RNA polymerase-rRNA promoter complex to a more stable form is the determining step. This step is salt-sensitive; ppGpp acts on this "isomerization". 相似文献
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10.
No reports on isolating RNA from carbohydrate-rich wheat seeds have been published. Because of the presence of carbohydrates,
published protocols yield small amounts of poor quality RNA. Extracting seeds in a buffer (pH 9, 150 mM NaCl, 1% sarcosyl)
ensured maximum RNA solubility and the removal of most interfering substances. Extracted RNA was purified using a guanidine
hydrochloride-based buffer system. This protocol yields up to 148 μg of RNA from 100 mg of tissue in 3.5 h. An A260/A280 ratio of 1.85 indicates RNA purity. Isolated RNA was amenable to downstream applications such as differential display. The
developed method was extended to other carbohydrate-rich seeds, such as barley and maize, with success. 相似文献
11.
George H. Shimer Jr. A-Young M. Woody Robert W. Woody 《Biochimica et Biophysica Acta (BBA) - Gene Structure and Expression》1988,950(3)
The interaction of Escherichia coli RNA polymerase with poly[d(A-T)] and poly[d-(I-C)] was studied by difference absorption spectroscopy at temperatures, from 5 to 45°C in the absence and presence of Mg2+. The effect of KCl concentration, at a fixed temperature, was studied from 12.5 to 400 mM. Difference absorption experiments permitted calculation of the extent of DNA opening induced by RNA polymerase and estimation of the equilibrium constant associated with the isomerization from a closed to an open RNA polymerase-DNA complex. ΔH0 and ΔS0 for the closed-to-open transition with poly[d(A-T)] or poly[d(I-C)] complexed with RNA polymerase are significantly lower than the values associated with the helix-to-coil transition for the free polynucleotides. For the RNA polymerase complexes with poly[d(A-T)] and poly[d(I-C)] in 50 mM KCl, ΔH0 ≈ 15–16 kcal/mol (63–67 kJ/mol) and ΔS0 ≈ 50–57 cal/K per mol (209–239 J/K per mol). The presence of Mg2+ does not change these parameters appreciably for the RNA polymerase-poly[d(A-T)] complex, but for the RNA polymerase-poly[d(I-C)] complex in the presence of Mg2+, the ΔH0 and ΔS0 values are larger and temperature-dependent, with ΔH0 ≈ 22 kcal/mol (92 kJ/mol) and ΔS0 ≈ 72 cal/K per mol (approx. 300 J/K per mol) at 25°C, and ΔCp0 2 kcal/K per mol (approx. 8.3 kJ/K per mol). The circular dichroism (CD) changes observed for helix opening induced by RNA polymerase are qualitatively consistent with the thermally induced changes observed for the free polynucleotides, supporting the difference absorption method. The salt-dependent studies indicate that two monovalent cations are released upon helix opening. For poly[d(A-T)], the temperature-dependence of enzyme activity correlates well with the helix opening, implying this step to be the rate-determining step. In the case of poly[d(I-C)], the same is not true, and so the rate-determining step must be a process subsequent to helix opening. 相似文献
12.
四步法消除SYBR GreenⅠ实时定量RT-PCR中引物二聚体的影响 总被引:18,自引:0,他引:18
为建立一种新的SYBRgreenⅠ实时定量RT PCR方法 ,使之能够有效消除引物二聚体 (PDs)对实时定量结果的影响 .对RT PCR特异性扩增产物和PDs分别进行了凝胶电泳检测和熔解曲线分析 .依据PDs和扩增产物的熔解温度 (Tm)特点 ,在通用的三步法的延伸步骤之后 ,增加一个短暂的 (5s)恒温和荧光检测步骤 ,使这个步骤的温度高于PDs的Tm,但低于扩增产物的Tm,简称该法为四步法 .PDs的Tm 通常高于 72℃ ,但低于扩增产物的Tm .将四步法第四步的温度设置在高于PDs的Tm ,但低于扩增产物的Tm 时 ,四步法能够有效地消除PDs的影响 .对三步法和四步法SYBRgreenⅠ实时定量RT PCR进行了比较 ,发现三步法根本不能用于RNA的实时定量 ,而四步法能够实现包括低丰度RNA在内的RNA的定量 .选择Tm 值尽可能小的引物 ,使PDs与扩增产物Tm 值之间有足够的差距 ,将更有利于四步法的应用 ,并可成功地用于低丰度RNA的准确定量 相似文献
13.
Alan J. Slusarenko 《Plant Molecular Biology Reporter》1990,8(4):249-252
A procedure which avoids the use of phenol-chloroform and RNAase for the isolation of total DNA fromA. tumefaciens is described. Specific precipitation of protein by 2.5 M ammonium acetate is employed and much of the RNA is removed by an isopropanol
precipition step. The procedure yields easily restrictable, good quality DNA and is probably applicable to other Gramnegative
bacteria. 相似文献
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During testicular germ cell differentiation, the structure of nuclear chromatin dynamically changes. The following describes a method designed to preserve the three-dimensional chromatin arrangement of testicular germ cells found in mice; this method has been termed as the three-dimensional (3D) slide method. In this method, testicular tubules are directly treated with a permeabilization step that removes cytoplasmic material, followed by a fixation step that fixes nuclear materials. Tubules are then dissociated, the cell suspension is cytospun, and cells adhere to slides. This method improves sensitivity towards detection of subnuclear structures and is applicable for immunofluorescence, DNA, and RNA fluorescence in situ hybridization (FISH) and the combination of these detection methods. As an example of a possible application of the 3D slide method, a Cot-1 RNA FISH is shown to detect nascent RNAs. The 3D slide method will facilitate the detailed examination of spatial relationships between chromatin structure, DNA, and RNA during testicular germ cell differentiation. 相似文献
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Extraction of RNA from tissues containing high levels of procyanidins that bind RNA 总被引:29,自引:1,他引:28
Commonly used methods for extraction of RNA from plants are not effective for isolation of high quality RNA from the pigmented
seed coats of soybeans that produce procyanidins (tannins) during seed coat development. We demonstrate a significant modification
of the phenol-LiCl method that yields high quality RNA from a black seed coat variety. In this method, seed coat material
was ground in a buffer containing a high concentration of bovine serum albumin (100 mg BSA/50 mg of lyophilized seed coats)
to competitively inhibit proanthocyanidin binding. The presence of hydrated insoluble polyvinylpoly-pyrrolidone (PVPP) was
also necessary to bind proanthocyanidins and remove them from solution. Proteinase K was added to digest the remaining BSA,
and phenol extraction was used to remove both the proteins and small molecular weight complexes formed by BSA and proanthocyanidins.
After LiCl and ethanol precipitations, the RNA quality was examined by UV absorbance spectra, gel electrophoresis, and hybridization.
Using this method, good quality RNA can be extracted from pigmented seed coats of soybean varieties that are homozygous for
the recessivei allele and also contain the dominantT gene that results in production of procyanidins in the seed coat. The method is also effective for tissues from other plant
species that contain abundant polyphenolic compounds. 相似文献
18.
Chris S. Jones Pietro P. M. Iannetta Mary Woodhead Howard V. Davies Ronnie J. McNicol Mark A. Taylor 《Molecular biotechnology》1997,8(3):219-221
Previous attempts to extract high-quality, total RNA from raspberry (Rubus idaeus) fruits using published protocols have proven to be unsuccessful. Even the use of protocols developed for the extraction
of RNA from other fruit tissue has resulted in low yields (1) or the isolation of degraded RNA (2). Here, we report on the development of a quick and simple method of extracting total RNA from raspberry fruit. Using this
method, high yields of good quality, undegraded RNA were obtained from fruit at all stages of ripening. The RNA is of sufficient
quality for northern analysis and cDNA library construction. 相似文献
19.
Kristel Eggermont Inge J. Goderis Willem F. Broekaert 《Plant Molecular Biology Reporter》1996,14(3):273-279
We describe here a reliable high-throughput method for extraction of RNA from fresh or frozen plant tissue that obviates laborious
and time-consuming homogenisation by mortar and pestle. The method is based on homogenisation by high-speed reciprocal shaking
in presence of a mixture of inexpensive abrasive materials; i.e., quartz sand and glass beads. After homogenisation, the method
follows a standard procedure for RNA extraction by phenol/LiCl. Yield and quality of RNA obtained by homogenisation with the
sand/glass bead mix are identical to those obtained by mortar and pestle. 相似文献