共查询到20条相似文献,搜索用时 140 毫秒
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We have developed a simple and rapid procedure for the isolation of total RNA from small amounts of adipose tissue. Using this method, it is possible to obtain quantitative recovery of RNA from less than 300 mg of adipose tissue, with an average yield of 70 micrograms of RNA per gram of adipose tissue. Northern blot analysis of rat epididymal adipose tissue RNA samples was performed using a beta-actin probe and demonstrated that intact total RNA had been isolated. The procedure has been adapted for use in 1.5-ml microcentrifuge Eppendorf tubes, providing a convenient and inexpensive method for the reproducible recovery of intact RNA from sparse samples of adipose tissue. 相似文献
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A procedure for the small-scale isolation of plant RNA suitable for RNA blot analysis 总被引:31,自引:0,他引:31
A small-scale method for the isolation of total RNA from plant tissue is described. The method provides RNA of suitable quantity and quality from 0.2 g fresh tissue for the detection of mRNA species by RNA blot analysis. The entire procedure is adapted to 1.5-ml microfuge tubes and takes less than 5 h. This method is well suited for the isolation of RNA from large numbers of samples or from samples of limited quantity. 相似文献
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目的用改进后的方法提取高质量的RNA,以此为模板,应用T-A克隆法克隆C57BL/6J小鼠周脂素基因编码区,对其进行测序验证,并与GenBank比对。方法在试剂说明书基础上,改进提取脂肪组织RNA的方法,从C57BL/6J附睾脂肪组织提取高质量的总RNA,用RT-PCR扩增出周脂素编码区基因,并将目的基因编码区克隆入pMD18-T载体中,转化E.coli JM109后,筛选阳性克隆,通过限制性内切酶酶切鉴定后,对其进行测序验证,并与GenBank比对。结果用改进后的方法成功提取出了高质量的总RNA,并且成功提取构建的重组载体中含有周脂素基因的全长序列,与GenBank公布的序列一致。结论改进的后的脂肪组织RNA提取方法是可行的,并获得周脂素基因的cDNA,为进一步研究其生物学功能奠定了基础。 相似文献
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Kiyohiro Hamatani Hidetaka Eguchi Keiko Takahashi Kazuaki Koyama Mayumi Mukai Reiko Ito Masataka Taga Wataru Yasui Kei Nakachi 《The journal of histochemistry and cytochemistry》2006,54(7):773-780
Recently, in addition to DNA, RNA extracted from archival tissue specimens has become an invaluable source of material for molecular biological analysis. Successful amplification with PCR/RT-PCR is problematic when using amplicons of short size due to degradation of DNA or RNA. We established an improved method for efficient RT-PCR amplification of RNA extracted from archival formalin-fixed, paraffin-embedded tissue by the elimination of RNA modification and the restoration of RNA template activity. Namely, the preheating in citrate buffer (pH 4.0) of RNA extracted from long-term preserved tissue specimens resulted in significantly increased efficiency of RT-PCR. 相似文献
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In the present study, we describe a method that we developed to isolate total RNA from porcine adipose tissue. This method entails homogenizing porcine adipose tissue in 10 ml of 4 M guanidium thiocyanate, 25 mM sodium citrate, 0.5% Sarcosyl, 0.1 M beta-mercaptoethanol, pH 7.0, and then performing two CHCl3 extractions to remove lipid before following the procedure described by P. Chomczynski and N. Sacchi (1987, Anal. Biochem. 162, 156-159). This modification improved the yield of RNA approximately threefold (yield was 88 +/- 7 micrograms total RNA/g of tissue) without affecting RNA quality. 相似文献
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Improved method for the isolation of RNA from plant tissues 总被引:149,自引:0,他引:149
A fast and efficient method for the isolation of RNA from plant tissues is described. Tuber tissue is homogenized in a guanidine hydrochloride-containing buffer followed by direct extraction with phenol/chloroform. The RNA is precipitated from the aqueous phase, washed with 3 M sodium acetate and 70% ethanol, and finally dissolved in water. The yield of RNA is up to 500 micrograms/g of tissue and several tests indicate intact and nondegraded RNA. This method can be adapted to a small-scale version by the use of 1.5-ml tubes, allowing rapid isolation of RNA from a larger number of samples. Finally, this method is of particular use for isolating RNA from tissues with a high polysaccharide and nuclease content such as wounded potato tubers. 相似文献
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Improved technique for isolating RNA from tobacco tissues 总被引:3,自引:0,他引:3
We have developed a much-improved method for isolating RNA from tobacco tissue. The novel component of the described RNA isolation
method is the addition of lithium chloride to the extraction buffer. Following that, the RNA was homogenized with phenol/chloroform
and precipitated in ethanol. This isolation technique provided highly reproducible and good quality RNA within 2 h. 相似文献
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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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We report a method for the isolation of total cellular RNA from mineralized or cartilaginous tissues. The procedure accommodates the large amount of hydroxyapatite and high buoyant density proteoglycans present in skeletal tissue samples, as well as the low cell density characteristic of these tissues. The procedure can be reliably used for processing a large number of small (100-800 mg) tissue samples. Tissues are homogenized in guanidine hydrochloride solution, then centrifuged at low speed, and filtered to remove the nonsolubilized extracellular matrix proteins. Subsequent high speed density gradient centrifugation produces a high yield of RNA (0.2-0.6 micrograms RNA/mg tissue) which is precipitated in a low pH sodium acetate solution. RNA extracted by this method has been analyzed for the expression of various genes by Northern blotting. In addition to mRNAs of bone- and cartilage-specific proteins, messenger RNA for growth factors, proto-oncogenes, and heat shock proteins can be detected. 相似文献
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An effective method for obtaining high-quality RNA from polysaccharide-rich hydrogels would be of great interest to biomaterialists and tissue engineers. Based on the similarities between polysaccharide-based hydrogels and plant tissues, we used a plant-specific RNA extraction kit to extract RNA from mammalian cells encapsulated in hydrogel scaffolds. The results indicate that this method can be reliably used in isolating high-purity RNA from polysaccharide hydrogels. 相似文献
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An efficient method for the isolation of RNA from cartilage is described. The difficulties in obtaining RNA from cartilage, a tissue of low cell density and high proteoglycan content, were overcome by making several modifications to the guanidine thiocyanate/cesium chloride method of RNA extraction. Cartilage tissue is frozen, crushed, and homogenized in a 4 M guanidine thiocyanate lysis buffer. The RNA is then pelleted by ultracentrifugation through a cesium trifluoroacetate density gradient. The use of cesium trifluoroacetate, rather than cesium chloride, for density gradient centrifugation improves both the yield and purity of total RNA isolated from cartilage. The ultracentrifugation has been adapted to the Beckman TL100 tabletop centrifuge and is complete in 3 h. This fast, simple method produces high quality RNA, suitable for use in RNase protection assays, polymerase chain reaction analysis, and Northern analysis. This purification procedure may be applicable to other sources, from which RNA isolation is complicated by the presence of abundant cell wall or matrix components. 相似文献
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A simple method has been developed that enables reextraction of RNA from an RNA-cDNA mixture. The reextracted RNA was converted
to cDNA followed by polymerase chain reaction (PCR). Thus, cDNA synthesis (followed by PCR) was carried out two times on the
same source of RNA. The method has been applied to 40 RNA samples of diverse tissue origin with a success rate of 100%. Thus,
the method offers more versatile use of small but valuable RNA sources than currently possible. 相似文献
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非洲菊花瓣RNA提取方法的改进 总被引:32,自引:1,他引:31
利用异硫氰酸胍一步法和植物总RNA提取试剂盒(RNeasy Plant Mini Kit)提取非洲菊(Gerbera hybrida)花瓣中总RNA时存在多糖的干扰。实验中利用异硫氰酸胍一步叶提取总RNA,在RNA沉淀后,再次加入适量变性液,冻融2次,再加热至45℃使RNA完全溶解;试剂盒提取时,适当延长各提取步骤的离心时间,并增加DEPC H2O溶解RNA的时间。通过以上操作方法的改进可排除多糖的干扰,得到高质量的总RNA。为进一步利用Northern杂交技术研究非洲菊中花色素苷基因的表达,进行花色改良提供了方便、有效的实验手段。 相似文献