十字花科植物种子低分子RNA提取方法比较

非编码RNA不翻译成蛋白质,它们通过转录、转录后及翻译水平调控靶基因表达,在植物生长发育及逆境胁迫中发挥功能。目前,大量种子萌发期特异表达的非编码RNA (Non-coding RNA)已被发现,高效提取种子低分子RNA是对其进行研究的关键。本研究将介绍一种改良SDS RNA提取方法,并与Trizol、CTAB法、RNA提取试剂盒进行比较。结果表明:这种方法可以高效提取用于Northern blotting、RT-PCR等分子生物学分析的十字花科植物种子低分子RNA。改良SDS RNA提取方法为种子非编码RNA研究、种子萌发生理及分子育种研究提供了帮助。     

三七总RNA提取方法的对比研究

比较利用改进的异硫氰酸胍一步法、异硫氰酸胍高盐法、CTAB法和Thomas’RNA提取法等4种方法提取三七根茎2个部位总RNA的可行性。结果表明,改进的异硫氰酸胍一步法和异硫氰酸胍高盐法能有效地抑制酚类物质、多糖及皂苷等次级代谢产物对总RNA的影响,可从三七根茎中获得质量高、完整性好的总RNA。RT—PCR分析显示提取的总RNA具有反转录活性。这2种方法具有快速、简单、有效的特点。     

干种子高质量总RNA的快速提取方法

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。     

一种提取高质量油菜种子和种皮RNA的方法

提取高质量的RNA是从基因表达水平上研究油菜种子和种皮发育的必要条件。现有方法因为油菜种子脂肪、多酚和多糖,难以快速获得完整、高纯度的油菜种子总RNA。本试验针对油菜种子和种皮特点,利用苯酚-氯仿抽提后用无水乙醇沉淀RNA,建立了在油菜种子和种皮中快速提取高质量总RNA的提取方法,电泳分析表明28S rRNA亮度约为18S rRNA的2倍;紫外分光光度计检测A260/A280介于1.8～2.0之间。用该法分离的RNA,已成功用于RT-PCR、Northern blot分析和基因全长的克隆等分子生物学研究。     

一种快速提取小麦叶片总RNA的方法

从植物组织中提取高质量的RNA是进行植物分子生物学研究的必要前提和关键.同种植物不同器官的组织由于组成分的差异,提取RNA的方法也存在不同的难点.在苯酚法和氯化锂沉淀法的基础上,改进并提出了一种适合小麦叶片总RNA的快速提取方法,消除了蛋白质、DNA、多糖、多酚等污染.该方法提取的小麦叶片总RNA,完整性好、纯度高,可用于RT-PCR、N orthern杂交、RACE等实验操作,而且简单经济、快速、实验结果稳定,重复性好,还适合富含多糖和脂质的植物组织总RNA的提取.     

富含多糖草莓果实总RNA提取方法的改进

以草莓果实为模式实验材料,将Chomczynski提出的常规RNA提取方法与Kenneth等提出的改进方法相结合,并做了进一步改进,建立了一种简单实用的从富舍多糖的植物材料中提取总RNA的方法。先利用冷的丙酮去除色素类物质,再利用乙二醇丁醚去除多糖,从而有效克服了从富含色素和多糖娄物质的植物材料中提取RNA的困难;获取的RNA样品在纯度和浓度上都可以满足PCR及Northern杂交等分子生物学实验的要求。     

仿刺参体壁总RNA提取方法的建立

目的:通过对TRIzol一步法进行改进,建立一种从富含胶原蛋白、多糖及色素的仿刺参体壁提取总RNA的有效方法。方法:样品在液氮中研磨并用TRIzol匀浆后再进行抽提;对TRIzol一步法提取的总RNA进行DNaseⅠ消化和酚氯仿抽提,用2.5mol/L的醋酸钾沉淀,并加入适量糖原(10mg/mL)与RNA共沉淀。结果:琼脂糖凝胶电泳和紫外分光光度法以及RT-PCR检测结果表明,改进的方法能够有效去除基因组DNA、蛋白、多糖及色素的污染,RNA的产率提高。结论:制备的总RNA纯度高,完整性好,能够满足mRNA差异显示RT-PCR等分子生物学研究的要求,是一种提取仿刺参体壁及其他富含黏多糖、胶原蛋白和色素的动物组织总RNA的有效方法。     

A Protocol for Extraction of High-quality RNA and DNA from Peanut Plant Tissues

Peanuts are an increasingly important global food source. However, until recently the lack of effective protocols for the extraction of nucleic acids has made molecular studies of peanut development and maturation difficult. Here, we describe a method to isolate high-quality RNA and DNA from peanut tissue and have successfully applied this method to peanut plant roots, stems, leaves, flowers, and seeds. Spectrophotometric analysis showed that the average yields of total RNA from 100 mg of peanut materials ranged from 24.52 to 74.6 μg, and those of genomic DNA from the same tissues ranged from 23.47 to 57.68 μg. Using this protocol, we obtained OD260/280 values between 1.9 and 2.0 and isolated RNA which could be reverse transcribed in a manner suitable for RT-qPCR and expression analysis. In addition, genomic DNA isolated using this method produced reliable restriction enzyme digestion patterns and could be used for Southern blot hybridization.     

An effective, rapid and simple method for total RNA extraction from bacteria and yeast

In this work, we describe a rapid and simple method for total RNA extraction from bacteria and yeast. The method allows for the acquirement of high RNA yields while avoiding the use of phenol or other toxic reagents and is less expensive than other methods previously described. The extracted RNA is suitable for applications such as RT-PCR, Northern blot hybridization and low molecular weight RNA (LMW RNA) electrophoresis.     

An optimized preparation method to obtain high-quality RNA from dry sunflower seeds

In an attempt to isolate high-quality, intact total RNA from sunflower (Helianthus annuus) seeds for investigation of the molecular mechanisms of mutations, we tested various procedures, using kits, including RNAiso Plus, RNAiso Plus+RNAiso-mate for Plant Tissue, Trizol, and the Qi method, but no high-quality total RNA of high integrity was obtained with any of these methods, probably due to the high content of polyphenols, polysaccharides, and secondary metabolites in mature sunflower seeds. Modifications were made to the Qi method. To avoid polyphenol oxidation, frozen dry seeds free of the seedcase were ground in a mortar with an equal amount of PVP30, and the fine ground powder was transferred to an extraction buffer with 2% PVP30 (w/v), 5% β-mercaptoethanol (v/v) and LiCl (8 M). A sample homogenate was extracted with chloroform prior to acidic phenol-chloroform extraction. The total RNA was precipitated with 1/4 volume of NaAc and 2 volumes of absolute ethanol to prevent contamination by polysaccharides. The yield of total RNA was 29.95 μg/100 mg husked dry seeds; the ratios of A260/A230 and A260/A280 were 2.44 and 2.09, respectively. Electrophoretic analysis clearly showed 28S and 18S ribosomal RNA bands. Using the extracted RNA, a fragment of the actin gene was successfully expressed by RT-PCR. This modified protocol is suitable for isolating high-quality total RNA from sunflower seeds for molecular research.     

An Improved Protocol for the Isolation of RNA from Roots of Tea (Camellia sinensis (L.) O. Kuntze)

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.     

Microbial gene expression in soil: methods, applications and challenges

About 99% of soil microorganisms are unculturable. However, advances in molecular biology techniques allow for the analysis of living microorganisms. With the advent of new technologies and the optimization of previous methods, various approaches to studying gene expression are expanding the field of microbiology and molecular biology. Methods used for RNA extraction, DNA microarrays, real-time PCR, competitive RT-PCR, stable isotope probing and the use of reporter genes provide methods for detecting and quantifying gene expression. Through the use of these methods, researchers can study the influence of soil environmental factors such as nutrients, oxygen status, pH, pollutants, agro-chemicals, moisture and temperature on gene expression and some of the mechanisms involved in the responses of cells to their environment. This review will also address information gaps in bacterial gene expression in soil and possible future research to develop an understanding of microbial activities in soil environments.     

Small-sample total RNA purification: laser capture microdissection and cultured cell applications.

Gene expression studies require analysis of RNA, but isolation of total RNA from very small samples by traditional methods can be difficult and inefficient. The Absolutely RNA microprep kit provides a convenient method for isolating total RNA from small numbers of cells such as those harvested by laser capture microdissection (LCM). The protocol includes binding of RNA to a solid support, thus eliminating the need for organic extraction and alcohol precipitation. DNase digestion on the solid support reduces or eliminates DNA contamination and minimizes RNA handling. Efficient washing removes contaminants, and elution in a small volume of buffer results in high-purity RNA at a concentration appropriate for demanding applications such as RT-PCR. RNA isolated from as few as 200 laser capture microdissected brain tumor cells resulted in detection of low, medium, and highly expressed genes by conventional and real-time RT-PCR.     

Supercritical carbon dioxide extraction of colchicine and related alkaloids from seeds of Colchicum autumnale L

A method for the extraction of the alkaloids colchicine, 3-demethylcolchicine and colchicoside from seeds of Colchicum autumnale by supercritical carbon dioxide has been established. Several parameters such as pressure, temperature, percentage of modifier and extraction time have been examined. Two extraction steps with constant carbon dioxide density (0.90 g/mL) and flux (1.5 mL/min) were required to extract the alkaloids in 110 min using 3% methanol as modifier. The quantitative determination of the alkaloids was performed by HPLC; the percentages of recovery were higher than 98% for the three alkaloids. This extraction procedure was compared with a conventional method involving maceration and sonication, and the same levels of alkaloids were obtained in each case. The supercritical carbon dioxide method is, however, very efficient, more rapid and more environmentally friendly than conventional methods.     

改良的大鼠脑组织总RNA抽提方法

目的:建立一种简单方便、结果稳定的抽提大鼠脑组织总RNA的方法。方法:在传统一步法抽提动物组织总RNA的基础上,通过增加抽提步骤使总RNA与细胞DNA、蛋白质、细胞残片等干扰总RNA质量的杂质有效分离。结果:建立了可以快速、稳定地获得高纯度、未被降解的大鼠脑组织总RNA的方法。结论:采用改良的方法抽提到的总RNA比用传统的一步法得到的总RNA的完整性和均一性好,可直接应用于分子生物学操作。