共查询到20条相似文献,搜索用时 15 毫秒
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High-quality RNA is important in studying gene expression. This report describes an improved method for isolating intact purified
RNA from dehydrated organs of chili pepper plants. Common RNA extraction protocols have produced poor yields because dehydrated
leaves accumulate polysaccharides and RNases. Our protocol is based on a guanidine thiocyanate extraction combined with additional
purification steps using butanol and the ionic detergent CTAB (cetyltrimethylammonium bromide). Using this protocol, RNA yields
ranged from 40–70 μg of total RNA per 200 mg of fresh tissue. This method can be adapted to large-scale isolations, allowing
the recovery of larger amounts of intact RNA (up to 250 μg per gram of fresh tissue). 相似文献
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Here we present a protocol for the extraction of RNA from Dictyostelium discoideum. Dictyostelium is a social amoeba that undergoes a basic developmental program, and therefore analysis of RNA levels over a time course is a commonly used technique. This procedure is similar to other guanidine thiocyanate-based methods; however, it has been adjusted because of the large quantities of carbohydrate and nucleases found in Dictyostelium cells. After cell lysis and phenol:chloroform extraction, the resulting high-quality RNA isolated with the described protocol allows the molecular genetic analysis of wild-type and genetically modified cells. The purified RNA can be used for analyses such as northern blotting, RT-PCR and microarrays. This procedure requires approximately 2 h to complete. 相似文献
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Purification of high-quality RNA from different strawberry tissues is often affected by the presence of high levels of contamination
by polysaccharides and phenolic compounds. With the protocol detailed here we describe for the first time total RNA purification
from petiole tissue. Treating the plants used as source of material with short-day light regime prior the extraction we are
able to obtain RNA suitable for further applications such as in vitro translation, RT-PCR, and RNA blot analysis. The yield
of total RNA extraction is significantly enhanced when tissue from plants grown under short-day photoperiodic condition is
used compared with that taken from plants grown under long day photoperiod. 相似文献
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干种子高质量总RNA的快速提取方法 总被引:1,自引:0,他引:1
高效快速提取高质量的种子RNA是种子分子生物学研究的基础。现有的提取方法难以高效快速地从种子中得到高质量的总RNA。本试验有机地将改进SDS法和异硫氰酸胍法相结合,采用改进的酸性SDS提取液、不溶性PVPP(聚乙烯聚吡咯烷酮)阻止酚类氧化、KAc去除多糖、异丙醇沉淀RNA,可以高效地从0.01~0.1g水稻、大豆、蚕豆、芸豆、花生等干种子中提取到高质量总RNA。此法提取的总RNA,能够满足分子生物学研究的要求,可以进行反转录和RT-PCR反应,用于基因表达研究,并为从具相似成分的其他物种干种子提取总RNA提供参考方法。 相似文献
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Elizabeth J. White Mauritz Venter Dr. Nicholas F. Hiten Johan T. Burger 《Biotechnology journal》2008,3(11):1424-1428
A wide range of plant RNA extraction methods are available; however, many of these are limited in their application for a diverse range of plant species. With special emphasis on robustness and versatility, we have improved the cetyltrimethylammonium bromide (CTAB) method and isolated high-quality RNA from 16 different plant species. The major modifications made to the protocol described here were a reduction of sample treatment steps and an increase in β-mercaptoethanol concentration (to 3%) resulting in a robust, rapid and reproducible plant RNA extraction protocol that can be used for a broad range of plant species and tissue types. 相似文献
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Isolating RNA from dry conifer seeds can be difficult because of a number of interfering compounds present in seeds. We describe
a protocol for total RNA isolation from black spruce dry seeds, which is an adaptation of a method used for mouse myeloma
tissues. The extraction relies on selective precipitation of RNA by using lithium chloride. 相似文献
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Many plant RNA isolation techniques aim to prevent contamination by means of secondary phenolics, carbohydrates, RNase, and
other chemicals. However, when applied in our laboratory to the isolation of RNA fromRumex obtusifolius, these protocols failed to produce good quality RNA. A major problem was contamination of the RNA samples with the secondary
metabolite oxalate. The relative quantities of guanidine isothiocyanate extraction buffer to plant tissue used in the protocol
had significant effects on oxalate contamination. An increase in extraction buffer, from 1.5 mL in the original method to
15 mL per 200–300 mg of tissue in our protocol, removed the oxalate from the RNA. This RNA was of a good quality and was suitable
for molecular biology applications. 相似文献
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An improved method for extracting proteins from leaf tissues of Aloe vera L., a recalcitrant plant species, for proteomic analysis is presented. In this protocol, the following critical components
are included. A washing step is added prior to homogenization of the tissue to eliminate contaminants, and a concentrated
2× extraction buffer (pH 7.5) is used to increase protein yield. Compared to classical trichloroacetic acid–acetone and phenol
extraction methods, this novel protocol has yielded two-dimensional electrophoresis gels with minimal (if any) streaking and
provided high-quality protein samples. This protocol is expected to be applicable to other recalcitrant plant tissues. 相似文献
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Anna Jaeschke Nicholas R. Harvey Mikhail Tsurkan Carsten Werner Lyn R. Griffiths Larisa M. Haupt Laura J. Bray 《Open biology》2021,11(6)
Three-dimensional (3D) cell culture models that provide a biologically relevant microenvironment are imperative to investigate cell–cell and cell–matrix interactions in vitro. Semi-synthetic star-shaped poly(ethylene glycol) (starPEG)–heparin hydrogels are widely used for 3D cell culture due to their highly tuneable biochemical and biomechanical properties. Changes in gene expression levels are commonly used as a measure of cellular responses. However, the isolation of high-quality RNA presents a challenge as contamination of the RNA with hydrogel residue, such as polymer or glycosaminoglycan fragments, can impact template quality and quantity, limiting effective gene expression analyses. Here, we compare two protocols for the extraction of high-quality RNA from starPEG–heparin hydrogels and assess three subsequent purification techniques. Removal of hydrogel residue by centrifugation was found to be essential for obtaining high-quality RNA in both isolation methods. However, purification of the RNA did not result in further improvements in RNA quality. Furthermore, we show the suitability of the extracted RNA for cDNA synthesis of three endogenous control genes confirmed via quantitative polymerase chain reaction (qPCR). The methods and techniques shown can be tailored for other hydrogel models based on natural or semi-synthetic materials to provide robust templates for all gene expression analyses. 相似文献
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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
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Sina Atashpaz Sajjad Khani Abolfazl Barzegari Jaleh Barar Sepideh Zununi Vahed Reza Azarbaijani Yadollah Omidi 《Microbiology》2010,79(4):538-542
The intactness of DNA is the keystone of genome-based clinical investigations, where rapid molecular detection of life-threatening
bacteria is largely dependent on the isolation of high-quality DNA. Various protocols have been so far developed for genomic
DNA isolation from bacteria, most of which have been claimed to be reproducible with relatively good yields of high-quality
DNA. Nonetheless, they are not fully applicable to various types of bacteria, their processing cost is relatively high, and
some toxic reagents are used. The routine protocols for DNA extraction appear to be sensitive to species diversity, and may
fail to produce high-quality DNA from different species. Such protocols remain time-consuming and tedious, thus to resolve
some of these impediments, we report development of a very simple, rapid, and high-throughput protocol for extracting of high-quality
DNA from different bacterial species. Based upon our protocol, interfering phenolic compounds were removed from extraction
using polyvinylpyrrolidone (PVP) and RNA contamination was precipitated using LiCl. The UV spectrophotometry and gel electrophoresis
analysis resulted in high A
260/A
280 ratio (>1.8) with high intactness of DNA. Subsequent evaluations were performed using some quality-dependent techniques (e.g.,
RAPD marker and restriction digestions). The isolated DNA from 9 different bacterial species confirmed the accuracy of this
protocol which requires no enzymatic processing and accordingly its low-cost making it an appropriate method for large-scale
DNA isolation from various bacterial species. 相似文献
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Vanessa Dos Reis Falcão Angela Pedroso Tonon Mariana Cabral Oliveira Pio Colepicolo 《Journal of applied phycology》2008,20(1):9-12
RNA isolation is essential to study gene expression at the molecular level. However, RNA isolation is difficult in organisms
(plants and algae) that contain large amounts of polysaccharides, which co-precipitate with RNA. Currently, there is no commercial
kit available, specifically for the isolation of high-quality RNA from these organisms. Furthermore, because of the large
amounts of polysaccharides, the common protocols for RNA isolation usually result in poor yields when applied to algae. Here
we describe a simple method for RNA isolation from the marine red macroalga Gracilaria tenuistipitata var. liui Zhang et Xia (Rhodophyta), which can be applied to other plants and algae. 相似文献