共查询到20条相似文献,搜索用时 15 毫秒
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Juan P. Valenzuela-Avendaño Iván A. Estrada Mota Gabriel Lizama Uc Ramón Souza Perera Elisa M. Valenzuela-Soto José J. Zúñiga Aguilar 《Plant Molecular Biology Reporter》2005,23(2):199-200
Isolation of high-quality RNA is a necessary step in gene expression analysis. Although many methods can be used to isolate
RNA from plants where contamination of preparations with complex carbohydrates or phenolic compounds is a problem, the application
of these methods toSelaginella lepidophylla tissues has failed to obtain good-quality RNA. Here we introduce 2 modifications to the method developed by Chomczynski and
Sacchi (1987), generating a simple and rapid method that allows the isolation of intact RNA fromS. lepidophylla-dehydrated tissues. Although the introduced modifications are not new, their addition proved to be decisive for success in
RNA isolation. Quality of the RNA obtained was evaluated by electrophoresis in agarose and by 3 different PCR-based techniques—RT-PCR,
RNA differential display, and synthesis of a cDNA library. 相似文献
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Isolation of Functional RNA from Periderm Tissue of Potato Tubers and Sweet Potato Storage Roots 总被引:3,自引:0,他引:3
David L. Scott Jr. Clarence W. Clark Kenneth L. Deahl Channapatna S. Prakash 《Plant Molecular Biology Reporter》1998,16(1):3-8
A reliable and efficient protocol is given for the isolation of mRNA from the periderm of potato tubers and sweet potato storage roots. The method relies on a urea-based lysis buffer and lithium chloride to concentrate total RNA away from most of the cytoplasmic components and to prevent oxidation of phenolic complexes. To enhance the physical separation of the RNA from other macromolecular components, the RNA fraction was incubated in the presence of the cationic surfactant Catrimox-14. Poly(A)+ mRNA was separated from total RNA and other contaminants by using Promega's MagneSphere technology. The mRNA was suitable for cDNA library construction and RNA fingerprinting. 相似文献
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C. Huang J. F. Picimbon H. Q. Li Z. Li Q. Liu W. Liu 《Russian Journal of Plant Physiology》2012,59(1):129-133
Most of conventional RNA extraction methods failed to extract highly pure and integral RNA from peanut seeds because peanut
seeds are extremely rich in lipids, proteins, polysaccharides, and phenolic compounds. Here, we describe a new method, named
Peanut Improved Modified RNA extraction method (PIMRNAext), using SDS, Tris-saturated phenol, NaCl, and sarkosyl during the
extraction process, which are particularly successful for total RNA extraction from lipid- and polysaccharide-rich materials.
The proposed PIMRNAext method is simple and fast. It requires only conventional reagents and is completed within 2 h. Using
PIMRNAext gives very good yields of high quality peanut RNA. This method is about ten times more efficient than conventional
methods, and the RNA produced by it is compatible with further molecular biology experiments, such as RT-PCR. We propose to
use the PIMRNAext method to extract RNA from peanuts and peanut-like plant species not only for RT-PCR, but also for most
molecular biology techniques that need copies of pure RNA, such as microarray or cDNA library construction. 相似文献
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Laura Jaakola Anna Maria Pirttilä Minna Halonen Anja Hohtola 《Molecular biotechnology》2001,19(2):201-203
A simple and efficient method is described for isolating high quality RNA from bilberry fruit. The procedure is based on the
use of hexadecyltrimethyl ammonium bromide (CTAB), polyvinylpyrrolidone (PVP), and β-mercaptoethanol in an extraction buffer
in order to eliminate the polysaccharides and prevent the oxidation of phenolic compounds. This method is a modification of
the one described for pine trees, and yields high-quality RNA suitable for cDNA based methodologies. This method is applicable
for a variety of plant tissues. 相似文献
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Covas MI de la Torre K Farré-Albaladejo M Kaikkonen J Fitó M López-Sabater C Pujadas-Bastardes MA Joglar J Weinbrenner T Lamuela-Raventós RM de la Torre R 《Free radical biology & medicine》2006,40(4):608-616
Olive oil phenolic compounds are potent antioxidants in vitro, but evidence for antioxidant action in vivo is controversial. We examined the role of the phenolic compounds from olive oil on postprandial oxidative stress and LDL antioxidant content. Oral fat loads of 40 mL of similar olive oils, but with high (366 mg/kg), moderate (164 mg/kg), and low (2.7 mg/kg) phenolic content, were administered to 12 healthy male volunteers in a cross-over study design after a washout period in which a strict antioxidant diet was followed. Tyrosol and hydroxytyrosol, phenolic compounds of olive oil, were dose-dependently absorbed (p<0.001). Total phenolic compounds in LDL increased at postprandial state in a direct relationship with the phenolic compounds content of the olive oil ingested (p<0.05). Plasma concentrations of tyrosol, hydroxytyrosol, and 3-O-methyl-hydroxytyrosol directly correlated with changes in the total phenolic compounds content of the LDL after the high phenolic compounds content olive oil ingestion. A 40 mL dose of olive oil promoted a postprandial oxidative stress, the degree of LDL oxidation being lower as the phenolic content of the olive oil administered increases. In conclusion, olive oil phenolic content seems to modulate the LDL phenolic content and the postprandial oxidative stress promoted by 40 mL olive oil ingestion in humans. 相似文献
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Kolosova N Miller B Ralph S Ellis BE Douglas C Ritland K Bohlmann J 《BioTechniques》2004,36(5):821-824
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黄檗(Phellodendron amuranse)叶片总RNA提取方法研究 总被引:2,自引:0,他引:2
以黄檗(Phellodendron amuranseRupr.)叶片为材料,分别利用改进的盐酸胍法、Trizol法、CTAB法提取黄檗叶片总RNA,通过RNA产率、纯度、电泳图谱等分析,确立了1种从黄檗叶片中快速分离总RNA的方法。研究结果表明,改进盐酸胍法所提取的总RNA的A260/A280为1.928,28S和18S条带清晰谱图完整性好,而且具有产率高、时间短、成本低的特点,所提取的总RNA适用于mRNA分离、cDNA文库的建立、Northern杂交等分析。 相似文献
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Tea, a beverage crop, is a rich source of polyphenols and polysaccharides which greatly attribute to its importance. However, oxidation and precipitation of these compounds during nucleic acids extraction is a limitation to molecular biology and genomic studies. On isolation of total RNA from root tissue using established protocols, difficulties were encountered in terms of purity and quantity of isolated RNA or some of the methods were time-consuming and also yields were low. The present communication combines a phenol-based RNA isolation protocol with a cetyltrimethylammonium bromide-based procedure with appropriate modifications. This protocol successfully isolated RNA from tap root tissue in 2-3 h as compared with 16 h reported by the previous method. Also, RNA yield was higher by more than fourfold. The RNA isolated by this protocol was successfully used for downstream applications such as RT-PCR and the construction of suppression subtractive hybridization library. The developed protocol worked well with other plant tissue with high polyphenols and polysaccharides contents. 相似文献
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Rafael da Silva Messias Vanessa Galli Julieti Huch Buss Joyce Moura Borowski Leonardo Nora Sérgio Delmar dos Anjos e Silva 《Preparative biochemistry & biotechnology》2013,43(7):697-707
The study of gene expression in maize varieties represents a powerful tool aiming to increase vitamin A precursors. However, the isolation of RNA from different maize varieties is challenging because these varieties show different levels of polysaccharides, and most methods available for RNA isolation are inappropriate for grain samples. The polysaccharides co-purify and co-precipitate with RNA during isolation, resulting in low-quality RNA, compromising the use of RNA in subsequent applications. Thus, a cetyltrimethylammonium bromide (CTAB)-based method was adapted in this study and compared with six methods for RNA isolation, including commercial reagents and RNA and DNA isolation kits, in order to identify the most appropriate for maize grains from different varieties. Most of the methods evaluated were considered inadequate due to limitations in terms of purity and/or quantity of the isolated RNA, which affected the efficiency of subsequent RT-qPCR analysis, resulting in nonamplification of β-carotene hydroxylase gene (HYD3) or high deviation among replicates. However, the CTAB modified method allowed the study to obtain intact RNA, with high quality and quantity, from 25 maize varieties. Furthermore, this RNA was successfully used to evaluate the expression of HYD3 gene by real-time qualitative polymerase chain reaction (RT-qPCR), and thus represents a simple, efficient, and low-cost strategy. 相似文献
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Efraim Lewinsohn Christopher L. Steele Rodney Croteau 《Plant Molecular Biology Reporter》1994,12(1):20-25
Stems of woody conifers contain high levels of polysaccharides and phenolic compounds that complicate the isolation of functional RNA from this highly lignified tissue. These difficulties were overcome by pulverizing the frozen tissue with a stainless-steel mortar and by effectively sequestering interfering phenolic compounds with vinylpyrrolidone polymers, thereby minimizing damage to nucleic acids during extraction. RNases were inhibited by aurintricarboxylic acid, and gelatinous polysaccharides were removed by inclusion of a 10% ethanol precipitation step. RNA was then isolated by precipitation with 33% isopropanol and ultracentrifugation onto a cushion of 5.7 M CsCl. Yields of 10 to 20 μg RNA/g FW were obtained from woody stems of several different gymnosperm species, including grand fir (Abies grandis), lodgepole pine (Pinus contorta), loblolly pine (Pinus taeda), Douglas fir (Pseudotsuga menziesii) and Pacific yew (Taxus brevifolia). The high quality of the RNA obtained was determined by UV-spectrophotometry, denaturing agarose gel electrophoresis, andin-vitro translation, and this material was used to construct cDNA libraries. 相似文献
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一种适用范围广的总RNA提取方法 总被引:23,自引:0,他引:23
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。 相似文献
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