共查询到20条相似文献,搜索用时 0 毫秒
1.
An efficient, simple, and small-scale procedure for isolating functional ribonucleic acid (RNA) was successfully applied to
many different tissues of grape and apple. These woody plants are rich in polyphenolic compounds and polysaccharides that
could impair the RNA extraction. The method chosen is based on the use of hot borate buffer at alkaline pH supplemented with
several adjuvants and followed by selective precipitations. Starting with only 0.4 g of fresh tissue and working with small
tubes (2 mL), we were able to obtain good yields of high-quality RNA suitable for further applications. The procedure can
be proposed for many applications, and it is particularly highly recommended when isolating RNA from a large number of samples. 相似文献
2.
RNA and DNA isolation from recalcitrant plant tissues. 总被引:9,自引:0,他引:9
3.
4.
Using silica particles to isolate total RNA from plant tissues recalcitrant to extraction in guanidine thiocyanate 总被引:1,自引:0,他引:1
The most commonly used protocol of the RNA isolation, the guanidine thiocyanate method, was unsuitable for recalcitrant plant tissues containing a large amount of storage proteins and secondary metabolites. We demonstrated that RNA could bind to the silica particles, which have been used successfully in DNA isolation from various sources, under a high concentration of NaCl in the presence of ethanol and sodium acetate. Based on this observation, an efficient, inexpensive, and highly reproducible technique, the acid phenol-silica method, was developed to isolate high-quality RNAs from various plant tissues recalcitrant to extraction in guanidine thiocyanate. 相似文献
5.
6.
High-quality total RNA was extracted using a cethyltrimethylammonium bromide-containing buffer followed by an acid guanidium thiocyanate-phenol-chloroform treatment from recalcitrant plant tissues such as tree leaves (pine, Norway spruce, ginkgo, Japanese cedar, rose), flowers (rose, Lotus japonicus) and storage tissues (seeds of Lotus japonicus and rice, sweet potato tuber, banana fruit). This protocol greatly reduced the time required for RNA extraction. 相似文献
7.
A method for the isolation of RNA from different tissues of trees (seedlings, saplings, and adult trees) is described. Using this procedure it is possible to remove large amounts of disturbing polyphenolic compounds from nucleic acids. The method involves an acetone treatment of the freeze-dried and powdered plant material, the use of high salt concentrations in the extraction buffer and an aqueous two-phase system. These steps were combined with the conventional phenol/chloroform extraction and CsCl centrifugation. The method has been successfully applied to the isolation and purification of RNA from pine (Pinus sylvestris L. and Pinus mugo Turr.), Norway spruce (Picea abies L.), and beech (Fagus sylvatica L.). The functional quality of RNA extracted by this procedure has been characterized by its uv spectrum, by agarose gel electrophoresis with ethidium bromide staining, Northern blot hybridization, and in vitro translation. 相似文献
8.
Specialized microbodies have previously been isolated and characterized from fatty seedling tissues (glyoxysomes) and leaves (leaf peroxisomes). We have now examined 11 other plant tissues, including tubers, fruits, roots, shoots, and petals, and find that all contain particulate catalase, a distinctive common enzyme component of microbodies. On linear sucrose gradients the catalase activity peaks sharply at a higher equilibrium density (1.20 to 1.25 gram per cm3 in the various tissues) than the mitochondria (1.17 to 1.20). Only small amounts of protein are recovered in the fractions containing catalase, although a definite band is visible in preparations from some tissues, e.g., potato. As in the preparations from castor bean endosperm and spinach leaves for which comparable data are provided, the distribution of glycolate oxidase and uricase follows closely that of catalase on the gradients. The preparations from potato lack glyoxylate reductase and the transaminases, typical enzymes of leaf peroxisomes, and the distinctive enzymes of glyoxysomes are missing. Nonspecialized microbodies with limited enzyme composition can thus be isolated from a variety of plant tissues. 相似文献
9.
This protocol describes a method for the extraction of DNA from elephant ivory. These techniques are being used to assign geographic origin to poached ivory by comparing the ivory genotype to a geographic-based gene frequency map, developed separately. The method has three components: ivory pulverization, decalcification and DNA extraction. Pulverization occurs in a freezer mill while the sample is deep frozen in liquid nitrogen, preventing degradation of DNA during the process. Decalcification involves repeated agitation of the sample in 0.5 M ethylenediaminetetraacetic acid over a 4-d period. Extraction follows a modified Qiagen protocol for the extraction of DNA from animal tissue. This method can be used on all forms of ivory. However, DNA recovery is highest when the outermost layer of the tusk, the cementum, is used. When applied to extract DNA from 11 samples, in duplicate, the entire protocol can be completed in 6 d, although much of this time consists of pause points that do not require effort. The protocol provides 0.8 +/- 0.11 ng microl(-1) (mean +/- s.e., n = 48) of DNA per sample. 相似文献
10.
A technique for the isolation of intact plastids from spinach (Spinacia oleracea) and pea (Pisum sativum) leaves, pea roots and castor bean (Ricinus communis) endosperm is described. This technique involves brief centrifugation of whole homogenates on density gradients. Intact plastids were located in the gradient by assaying for triose phosphate isomerase activity. Contamination of the plastic peak with mitochondria and microbodies was estimated by measurement of cytochrome oxidase and catalase, respectively. For three of the four tissues the level of contamination of the plastids by these organelles was 2% or less. The sedimentation behavior of microbodies from different tissues is discussed. 相似文献
11.
Isolation of total RNA from plant materials has been difficult, due to the presence of complex organic substances and the associated pigmentation. In fact, there is a dearth of standardized protocols for isolating total RNA from pollens. To find a simple and reliable method for isolating total RNA from pollen, four methods, viz. phenol/SDS (PS), guanidine HCl (GH), tri-reagent (TR), and modified SDS-betaME (SB) were tested with fresh pollen of Ricinus communis (procured at -70 degrees C) and pollen dried at 30-37 degrees C. The quality and quantity of RNA was superior for the material processed at -70 degrees C. SB gave the highest RNA yield (2.35 mg/g, OD260/280 >2.0), compared to other methods. The results obtained by the SB method were found to be comparable with the widely used tri-reagent method. This was validated with other pollens of Imperata cylindrica and Xanthium strumarium. The yield obtained from graded amounts of pollen was consistent with SB, compared to the TR method. The RNA isolated by SB gave good quality mRNA for synthesizing cDNA. The SDS-betaME method is simple, efficient, and uses less expensive reagents. Hence, we recommend the modified SDS-betaME method for isolating total RNA from pollens. 相似文献
12.
Iamkovaia TV Khomov VV Zagrebel'nyĭ SN Iamkovoĭ VI 《Prikladnaia biokhimiia i mikrobiologiia》2006,42(1):93-97
A simple method of production of total RNA from baker's yeast was developed. Total RNA was isolated from yeast (Saccharomyces cerevisiae) biomass using lysis with sodium dodecyl sulfate at 100 degrees C for 40-60 min and subsequent precipitation of the target product with 3 M NaCl. The preparation obtained was characterized in detail: yield of total RNA from 1 kg of pressed yeast, 9.25 g; optical density at 260 nm of 1 mg of RNA dissolved in 1 ml of water, 20.2 U; content of the acid-soluble fraction, 2.02%; and protein content, 1.8%. Total tRNA was isolated from total RNA by fractional precipitation with ethanol followed by gel filtration. 相似文献
13.
Mitochondrial DNA and RNA isolation from small amounts of potato tissue 总被引:11,自引:0,他引:11
Nunzia Scotti Teodoro Cardi Laurence Marechaldrouard 《Plant Molecular Biology Reporter》2001,19(1):67-67
We present a fast and simple protocol for purification of mitochondrial DNA and RNA from small amounts of potato tissue including
tubers, leaves, flowers, and flower buds. This method uses a high ionic strength medium to isolate mitochondria and extract
mitochondrial DNA and RNA from a single preparation and is easily adaptable to other plant species. The mitochondrial DNA
was not contaminated by plastid DNA, was fully restrictable and was successfully used for PCR, cloning and Southern analyses.
Similarly, the isolated mitochondrial RNA was not contaminated (flower buds) or only slightly contaminated (leaves) by plastid
RNA. RNA prepared according to our method was acceptable for northern and RT-PCR analyses. 相似文献
14.
In this report we present a strategy for generating a representative cDNA library from prohibitively low amounts of mRNA template. A defined DNA adapter, which carries an EcoRI site, is ligated to both ends of the products of a cDNA synthesis reaction. This allows low levels of cDNA to be amplified by a polymerase chain reaction. In studies with pg amounts of rabbit globin mRNA, the amplified cDNA product is shown to be full-length. Globin cDNA recombinants are positively identified in lambda gt10. The protocol should be widely applicable to mRNAs of low abundance, whose sequences have not been determined, and to limited samples from patients or animals. It may also be useful for generating representative libraries of low titer or variant viral sequences. 相似文献
15.
《Biochemical education》1999,27(2):110-113
Practical RNA experiments on RNA technology are rarely performed in intermediate and advanced biochemistry courses mainly due to the fact that ribonucleases are very active, widespread and stable. We describe here an easy practical experiment that, although it is not truly representative of RNA research, overcomes methodological difficulties and helps to integrate theoretical knowledge with practical findings. This experiment has been performed for two years in a Molecular Biology course using plants as the RNA source since they contain the three types of rRNA pairs that a cell can contain. By solving the Study Questions, the students understand the importance of the different types of RNA in the cell, assess the purity, yield and concentrations of their samples, and identify the origin of each band observed in agarose gel electrophoresis. 相似文献
16.
Rapid isolation of total RNA from mammalian tissues 总被引:2,自引:0,他引:2
A rapid procedure for the isolation of total RNA from small amounts of mammalian tissue (35 to 150 mg) is described. Tissues were homogenized in the presence of RNase inhibitors but in the absence of strong detergents. Contaminants were removed by phenol/chloroform extraction and Sephadex column chromatography. Total RNAs were precipitated with ethanol and sodium acetate. The RNAs isolated were intact and suitable for mRNA quantitation via Northern blot or slot-blot analyses. This procedure isolates total RNAs in high yield and purity, without CsCl ultracentrifugation, and is especially useful when mRNAs must be quantitated from many samples. 相似文献
17.
Tatjana Heinrich Stewart Washer John Marshall Michael G. K. Jones Robert H. Potter 《Molecular biotechnology》1997,8(1):7-12
A subtractive hybridization method is described that allows the generation of a subtractive gene library from small amounts
of plant or other eukaryotic tissues. The method uses paramagnetic oligo-dT beads to capture poly-adenylated mRNA and to synthesize
the complementary cDNA on a solid support. The use of magnetic beads facilitates the change of reaction buffers and the removal
of primers and minimizes yield losses. Subtracted material obtained from this method can either be cloned directly or used
to screen a specific library. 相似文献
18.
The Mexican marigold (Tagetes erecta) produces inflorescences of intense yellow color that contain high levels of xanthophylls, particularly lutein, which makes
it a suitable model for the study of carotenoid biosynthesis and regulation throughout the development of the inflorescences.
However, these studies require the recovery of total RNA from floral buds and inflorescences at different developmental stages,
each of which presents specific extraction problems. Four protocols were tested, but only through the modification of one
of them was it possible to obtain total RNA of sufficient quality and quantity to perform RT-PCR and Northern blots and to
construct a cDNA library. This article presents the modified protocol for the recovery of total RNA from carotenoid-rich plant
tissues. 相似文献
19.
20.