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1.
Background
The mocha mouse carries a spontaneous deletion in the Ap3d1 gene, encoding the delta 1 subunit of the adaptor related protein complex 3, (Ap3d1), and subsequently lack the expression of functional AP-3. This leads to a deficiency in vesicle transport and storage, which affects neurotransmitter vesicle turnover and release in the central nervous system. Since the genomic sequence of the Ap3d1 gene of mocha mouse is not known, precise mapping of the deletion as well as reliable genotyping protocols are lacking.Findings
We sequenced the Ap3d1 gene (HGNC GeneID: 8943) around the deletion site in the mocha mouse and revealed a 10639 bp deletion covering exon 2 to 6. Subsequently, new PCR primers were designed yielding a reliable genotyping protocol of both newborn and adult tissue. To examine the genotypes further, hippocampal neurons were cultured from mocha and control mice. Patch-clamp recordings showed that mocha neurons had a higher input resistance, and that autaptic EPSC in mocha cultures depressed faster and stronger as compared with control cultures.Conclusion
Our study reports the sequence of the deleted part of the Ap3d1 gene in mocha mice, as well as a reliable PCR-based genotyping protocol. We cultured hippocampal neurons from control and mocha mice, and found a difference in input resistance of the neurons, and in the synaptic short-term plasticity of glutamatergic autapses showing a larger synaptic depression than controls. The described procedures may be useful for the future utilization of the mocha mouse as a model of defective vesicle biogenesis. Importantly, as genotyping by eye color is complicated in newborn mice, the designed protocol is so fast and reliable that newborn mice could rapidly be genotyped and hippocampal neurons dissociated and cultured, which is normally best done at P0-P2. 相似文献2.
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RNASwift is an inexpensive, versatile method for the rapid extraction of RNA. Existing RNA extraction methods typically use hazardous chemicals including phenol, chloroform and formamide which are often difficult to completely remove from the extracted RNA. RNASwift uses sodium chloride and sodium dodecyl sulphate to lyse the cells and isolate the RNA from the abundant cellular components in conjunction with solid phase extraction or isopropanol precipitation to rapidly purify the RNA. Moreover, the purified RNA is directly compatible with downstream analysis. Using spectrophotometry in conjunction with ion pair reverse phase chromatography to analyse the extracted RNA, we show that RNASwift extracts and purifies RNA of higher quality and purity in comparison to alternative RNA extraction methods. The RNASwift method yields approximately 25 μg of RNA from only 108Escherichia coli cells. Furthermore, RNASwift is versatile; the same simple reagents can be used to rapidly extract RNA from a variety of different cells including bacterial, yeast and mammalian cells. In addition to the extraction of total RNA, the RNASwift method can also be used to extract double stranded RNA from genetically modified E. coli in higher yields compared to alternative methods. 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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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
6.
蔗糖合成酶(SuSy)是植物蔗糖代谢的关键酶,在植物生长发育过程中起着重要作用.为研究拟南芥中SUS3的功能,构建RNAi-SUS3干涉载体,通过农杆菌介导的真空渗透法转化拟南芥.筛选获得纯系转基因植株后,对AtSUS家族进行表达分析,利用环境扫描电子显微镜观察转基因植株表型,并对转基因拟南芥角果进行木质素组织化学染色以及透射电子显微镜检测.结果表明,RNA干涉技术能够抑制AtSUS3的表达,正常培养条件下该基因沉默后对拟南芥的表型没有显著影响,但可引起角果中AtSUS1,AtSUS2和AtSUS4表达代偿性增加,使转基因植株角果内果皮层细胞次生细胞壁增厚,木质化程度加深,同时果瓣厚度也有增加趋势.结果提示,转基因拟南芥角果的发育较野生型植株更为优先,AtSUS3基因沉默可能有利于角果的成熟. 相似文献
7.
Summary A simple and efficient procedure suitable for extraction of high-quality RNA from cultured conifer tissues, somatic embryos, zygotic embryos, needles, stem and root tissues was developed. It produced from 100 g up to 700 g total RNA per gram tissue dependent on the types of tissues used. RNA quality was estimated by spectrophotometry, agarose gel electrophoresis, in vitro translation of mRNA, cDNA synthesis and Northern blot analysis. The method also worked well with Arabidopsis thaliana and tobacco tissues.Abbreviations CTAB
cetyltrimethylammonium bromide
- DEPC
diethylpyrocarbonate
- PVP
polyvinylpyrrolidone
- SDS-PAGE
sodium dodecyl sulphate-polyacrylamide gel electrophoresis 相似文献
8.
A method for isolating total RNA from pear leaves 总被引:5,自引:0,他引:5
M. Malnoy J. P. Reynoird F. Mourgues E. Chevreau P. Simoneau 《Plant Molecular Biology Reporter》2001,19(1):69-69
Isolation of high quality RNA fromRosaceae species is particularly difficult. These plants contain considerable amounts of plant polyphenolic compounds and polysaccharides
that copurify with RNA, often rendering it unsuitable for either cDNA synthesis and/or hybridization in northern analyses.
We describe a method for RNA isolation from pear leaves that is modified from that of Manning (1990). The procedure includes
i) an extraction with phenol and PVPP, to remove proteins and polyphenols ii) two purifications by LiCl, with a 2-butoxyethanol
treatment between the LiCl steps. The method results in high quality RNA suitable for RT-PCR and northern blot experiments. 相似文献
9.
We combined the TRIzol method of nucleic acid extraction with QIAamp columns to achieve coextraction of RNA and genomic DNA from peripheral blood mononuclear cells (PBMCs) and biopsied skeletal muscle, both stored at −80 °C for many months. Total RNA was recovered from the upper aqueous phase of TRIzol. The interphase and organic phases were precipitated with ethanol, digested with proteinase K, and filtered through QIAamp MinElute columns to recover DNA. The combined protocol yielded excellent quality and quantity of nucleic acids from archived human PBMCs and muscle and may be easily adapted for other tissues. 相似文献
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《Bioscience, biotechnology, and biochemistry》2013,77(12):2674-2677
Double-stranded RNA (dsRNA) induces sequence-specific gene silencing in eukaryotes through a process known as RNA interference (RNAi). RNAi is now used as a powerful tool for functional genomics in many eukaryotes, including plants. We herein report a dsRNA-mediated transient RNAi assay system using protoplasts from Arabidopsis mesophyll cells and suspension-cultured cells (cell line T87). Introduction of dsRNA into protoplasts led to marked silencing of target transgenes. Our assay system would provide a convenient and efficient way to induce RNAi in protoplasts of the model plant Arabidopsis thaliana. 相似文献
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目的:建立一种从小鼠表皮组织提取高质量RNA的方法。方法:用热击法分离小鼠表皮,用TRIzol法提取RNA,用紫外分光光度计测定RNA的产率和纯度,用琼脂糖电泳和RT-PCR检测RNA的质量和完整性。结果:采用新方法提取的小鼠表皮总RNA,其D260nm/D280nm值为1.8~2.0,大于1.5,且RNA产率高于100μg/g;琼脂糖电泳出现5S、18S和28S等3条清晰的rRNA条带,而且28SrRNA条带的亮度约为18S的2倍;用新方法制备的总RNA可成功地用于RT-PCR实验。结论:采用热击法分离表皮并结合TRIzol法可提取到高质量、完整性好的小鼠表皮总RNA,并能用于相关的分子生物学实验。 相似文献
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Extraction of RNA from alfalfa pollen using conventional grinding devices resulted in low yields of degraded RNA; degradation
appeared to be related to the length of time required to break open the pollen grains with such devices. A glass syringe was
modified to provide the restricted, shearing environment necessary for rapidly rupturing this type of tissue. This apparatus
would be useful for extracting a variety of molecules from pollen of any species, but is particularly valuable for species
which produce low amounts of relatively small pollen grains. 相似文献
13.
Several protocols and commercial kits are used for the extraction of nucleic acids from different plant tissues. Although
there are several procedures available to remove sugars, which hinder the extraction of clean genomic DNA, there are few to
assist with extraction of RNA. Those presently used include precipitations with ethylene glycol monobutyl ether or lithium
chloride (LiCl), or centrifugation in cesium chloride (CsCl) gradients, but these generally either do not allow high recovery
of RNA, are time consuming, rely on hazardous chemicals or need special equipment. Here we present the use of the simple cation,
Ca2+, which has been tested and shown to be very efficient for the precipitation of high molecular weight pectic sugars during
RNA extraction. Results are presented for different plant tissues, especially tissues of peach and apple fruits at varying
ripening stages. 相似文献
14.
目的:建立一种从石蜡包埋组织提取高质量RNA的简便、经济的方法。方法:运用TRIzol法和改进的AGPC(酸-异硫氰酸胍-苯酚-氯仿)法等2种提取方法,从50例石蜡包埋组织中提取RNA,用紫外分光光度仪测定RNA的产率和纯度;用管家基因β-肌动蛋白做RT-PCR,检测RNA的质量。结果:TRIzol法提取RNA的成功率为96%(48/50),AGPC法为94%(47/50),2种方法所得RNA的吸光度值及得率在统计学上均无明显差异。结论:AGPC法和TRIzol法的提取效率相似,且提取费用只有TRIzol法的一半左右,是一种简便、经济、适合大量提取石蜡包埋组织中的RNA的方法,可用于临床。 相似文献
15.
Glenn C. Graham 《Plant Molecular Biology Reporter》1993,11(1):32-37
Carbohydrates, polyphenolic compounds, terpenoids and tannins interfere with the extraction of intact, uncontaminated total
RNA from conifers. A method for extraction of total RNA fromPinus radiata is described. This method uses cesium trifluoroacetate in the ultracentrifugal separation of RNA to overcome the problems
of co-purification of contaminating secondary metabolites. 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