何首乌总RNA提取方法的比较及改进

目的：探讨不同提取方法对提取何首乌总RNA的质量影响,寻找适于何首乌成熟叶组织RNA的提取方法。方法：以何首乌成熟叶组织为材料,采用SDS／酸酚法、常规CTAB法及改良的TRIzol试剂法分别进行实验,并对所提取RNA的质量进行验证。结果：采用3种方法都能提取出RNA,但质量差异较大。其中改良的TRIzol试剂法能有效抑制次生物质的影响,提取的RNA产量可达70-110μg／g,纯度高于其他2种方法,D260nm／D280nm值为1．85～1．97。结论：改良的TRIzol试剂法操作简便,提取的RNA完整性和纯度较高,可以满足下一步实验的要求。     

大青杨叶片总RNA的快速提取方法

目的:寻求快速提取大青杨叶片总RNA的方法。方法:分别用改良CTAB法、改良SDS法、改良TRIzol法及某公司总RNA提取试剂盒提取大青杨叶片总RNA,并用紫外光谱分析、凝胶电泳方法对提取的总RNA进行鉴定。结果:用改良CTAB法和改良TRIzol法能有效地去除蛋白及多糖,提取到的总RNA纯度高,D260nm/D280nm分别为2.05和1.78,RNA的完整程度优于试剂盒法。结论:改良CTAB法为大青杨叶片总RNA的最佳提取方法。     

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。     

Proteins isolated with TRIzol are compatible with two-dimensional electrophoresis and mass spectrometry analyses

TRIzol is used for RNA isolation but also permits protein recovery. We investigated whether proteins prepared with TRIzol were suitable for two-dimensional gel electrophoresis (2-DE) and matrix-assisted laser desorption/ionization mass spectrometry. Proteins from TRIzol-treated SH-SY5Y cells produced 2-DE spot patterns similar to those from an equivalent untreated sample. Subsequent identification of TRIzol-treated proteins using peptide mass fingerprinting was successful. TRIzol exposure altered neither the mass of myoglobin extracted from sodium dodecyl sulfate (SDS) gels nor the masses of myoglobin peptides produced by in-gel trypsin digestion. These findings suggest that proteins isolated with TRIzol remain amenable to proteomic analyses.     

草坪草总RNA几种提取方法的比较

分别采用DEPC水法、TRIzol法、CTAB-LiCl沉淀法、SDS-酚法、SDS提取液抽提法以及异硫氰酸胍法等6种方法提取草坪草总RNA,通过检测对各种方法的提取效果进行比较分析。结果表明,DEPC水法是最经济、操作最简单的一种方法,并且提取的RNA质量较好、可靠性高、易于大量制备,是提取草坪草RNA比较理想的方法之一,建议使用该方法。利用TRIzol法提取到的28S rRNA亮度约为18S rRNA的两倍,效果较好,操作简单,符合分子生物学实验要求。CTAB-LiCl沉淀法、SDS-酚法、SDS提取液抽提法也获得较高质量的RNA,但存在不同程度的DNA污染。异硫氰酸胍法提取RNA有明显的蛋白质污染,且RNA部分降解,此种方法不予采用。     

条斑紫菜丝状体总RNA提取方法比较

目的:为了获得质量较高的条斑紫菜丝状体总RNA,对几种常用提取方法进行研究。方法:以条斑紫菜自由丝状体为材料,比较了用异硫氰酸胍法、CTAB法、SDS/酚法、TRIzol法、RNAplant法提取的RNA的质量和纯度。结果:异硫氰酸胍法提取RNA的成本低,但纯度不高;CTAB法产率较小,且不能完全去除多糖或蛋白质;SDS/酚法未能获得完整的RNA;TRIzol法未能见到5SrRNA条带,且带有杂带;而RNAplant法提取RNA的质量好、纯度高、提取效率高,其D260nm/D280nm值为1.836,经逆转录得到的双链cDNA扩增产物长度在200bp以上。结论:实验结果表明RNAplant法更适于条斑紫菜丝状体总RNA的提取。     

A streamlined protocol for extracting RNA and genomic DNA from archived human blood and muscle

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.     

芍药花瓣总RNA的提取

用TRIzol法和改良的热硼酸盐法从芍药幼嫩花瓣中提取总RNA,总RNA的D260nm／D280nm值分别为1．803、1．974。琼脂糖电泳表明,用TRIzol法提取的RNA只有28S、18S2条带,带的亮度差,说明RNA有些降解;而用改良的热硼酸盐法提取的总RNA有28S、18S、5S3条带,带的亮度强,且28S是18S宽度的2倍左右,说明提取的RNA完整、未降解,可用于后续实验。改良的热硼酸盐法更适于从芍药花瓣中提取总RNA。     

Spermatozoa input concentrations and RNA isolation methods on RNA yield and quality in bull (Bos taurus)

Sperm RNA can be used to understand the past spermatogenic process, future successful fertilization, and embryo development. To study the sperm RNA composition and function, isolation of good quality RNA with sufficient quantity is essential. The objective of this study was to assess the influence of sperm input concentrations and RNA isolation methods on RNA yield and quality in bull sperm. The fresh semen samples from bulls (n = 6) were snap-frozen in liquid nitrogen and stored at −80 °C. The sperm RNA was isolated using membrane-based methods combined with TRIzol (RNeasy + TRIzol and PureLink + TRIzol) and conventional methods (TRIzol, Double TRIzol, and RNAzol RT). Based on fluorometric quantification, combined methods resulted in significantly (P < 0.05) higher total RNA yields (800–900 ng/30–40 × 10⁶) as compared with other methods and yielded 20 to 30 fg of RNA/spermatozoon. The quality of RNA isolated by membrane-based methods was superior to that isolated by conventional methods. The sperm RNA was observed to be intact as well as fragmented (50–2000 bp). The study revealed that the membrane-based methods with a cocktail of lysis solution and an optimal input concentration of 30 to 40 million sperm were optimal for maximum recovery of RNA from bull spermatozoa.     

Extraction of high-quality RNA from pancreatic tissues for gene expression studies

Extracting RNA from pancreatic tissue is notoriously challenging because of the organ's high RNase content. Standard methods using TriPure or TRIzol classically yield RNA of sufficient quality for routine gene expression analysis but not for microarray or deep sequencing analysis. Here we developed a simple method to extract high-quality RNA from mouse pancreas. Our method uses an RNase inhibitor and combines different protocols using guanidium thiocyanate–phenol extraction. It enables reproducible isolation of RNA with an RNA integrity number around 9.     

A rapid TRIzol-based two-step method for DNA-free RNA extraction from Arabidopsis siliques and dry seeds

Extraction of high-quality RNA from Arabidopsis seeds has been a challenge. Here we report a two-step TRIzol-based procedure for RNA extraction from Arabidopsis siliques and dry seeds. This procedure employs a modified, high pH (pH 9.5) extraction buffer. High pH plus the addition of either DTT or β-mercaptoethanol in the extraction buffer effectively inhibits RNase activity during the extraction, and removes most polysaccharides, polyphenols and other insoluble material. TRIzol reagent was subsequently used to purify the RNA. Using this procedure we isolated high-quality DNA-free RNA samples without DNase I treatment from Arabidopsis seeds or siliques in less than 3 h.     

应用非伤害性取样提取番鸭毛囊组织总RNA

目的:寻求一种从番鸭毛囊组织中高效提取总RNA的方法。方法:探讨了伤害性取样(剪切皮肤毛囊)、非伤害性取样(直接拔取毛囊)2种不同毛囊取样法对总RNA提取质量的影响,并对常用RNA提取方法TRIzol法中研磨和组织匀浆步骤细节稍加改进,琼脂糖电泳检测总RNA质量。结果:2种毛囊取样方法均能提取出高质量的总RNA,其28S、18S和5S条带清晰可见,无DNA污染。结论:非伤害性取样法可作为番鸭毛囊组织总RNA提取的适用取样方法。     

A rapid protocol for purification of total RNA for tissues collected from pigs at a slaughterhouse

Since RNA extraction is a crucial step in many molecular techniques, the protocols for sample collection and RNA purification need to be adapted to optimize their performance when samples are collected from animals at commercial facilities. Here we provide an RNA purification protocol for animal tissues collected from slaughterhouses. This protocol, modified from other techniques, uses TRIzol Reagent. Sample collection was performed wearing sterile gloves and facemasks, using sterile surgical instruments, and no longer than 8 min spent for each sample. A 0.9% sterile sodium chloride solution was used to wash the tissue before each sample collection. The whole process of RNA extraction was performed under cold environment and sterile conditions. This protocol produced good RNA yields (50 μg RNA per 100 mg tissue), good integrity and purity (Abs(260/280) from 1.8 to 2.0), from tissues such as liver, muscle, hypophysis, adipose tissue, and intestinal mucosa, in less than 2 h.     

Identification of extracellular miRNA in archived serum samples by next-generation sequencing from RNA extracted using multiple methods

miRNAs act as important regulators of gene expression by promoting mRNA degradation or by attenuating protein translation. Since miRNAs are stably expressed in bodily fluids, there is growing interest in profiling these miRNAs, as it is minimally invasive and cost-effective as a diagnostic matrix. A technical hurdle in studying miRNA dynamics is the ability to reliably extract miRNA as small sample volumes and low RNA abundance create challenges for extraction and downstream applications. The purpose of this study was to develop a pipeline for the recovery of miRNA using small volumes of archived serum samples. The RNA was extracted employing several widely utilized RNA isolation kits/methods with and without addition of a carrier. The small RNA library preparation was carried out using Illumina TruSeq small RNA kit and sequencing was carried out using Illumina platform. A fraction of five microliters of total RNA was used for library preparation as quantification is below the detection limit. We were able to profile miRNA levels in serum from all the methods tested. We found out that addition of nucleic acid based carrier molecules had higher numbers of processed reads but it did not enhance the mapping of any miRBase annotated sequences. However, some of the extraction procedures offer certain advantages: RNA extracted by TRIzol seemed to align to the miRBase best; extractions using TRIzol with carrier yielded higher miRNA-to-small RNA ratios. Nuclease free glycogen can be carrier of choice for miRNA sequencing. Our findings illustrate that miRNA extraction and quantification is influenced by the choice of methodologies. Addition of nucleic acid- based carrier molecules during extraction procedure is not a good choice when assaying miRNA using sequencing. The careful selection of an extraction method permits the archived serum samples to become valuable resources for high-throughput applications.     

Development of monoclonal antibody-coated immunomagnetic beads for separation and detection of norovirus (genogroup II) in faecal extract samples

Aims:  The aim of this study is to develop an RT-PCR assay combined with immunomagnetic beads (IMS/RT-PCR) coating monoclonal antibody (Mab) for separation and detection of norovirus (genogroup II) in faecal samples. We furthermore compare its detection limits with IMS/RT-PCR using polyclonal antibody (Pab) and the TRIzol extraction method followed by RT-PCR (TRIzol-RT-PCR).
Methods and Results:  Mab-coated beads and Pab-coated beads were added to a series of tenfold dilutions of faecal extract containing norovirus in 1 ml PBS. After incubation and collection, the RNA was released by heating from virus separated by beads. The tenfold dilutions of faecal were also extracted with TRIzol reagent. The RNA was used as the template for RT-PCR detection (primers: JV12–JV13). IMS/RT-PCR using Mab showed an endpoint in the 10⁻⁷ dilution and was 10² times more sensitive than IMS/RT-PCR using Pab and was at least 10³ times more sensitive than TRIzol-RT-PCR method.
Conclusions:  IMS/RT-PCR using Mab proved to be a more sensitive method of noroviruses (NVs) detection than IMS/RT-PCR using Pab and the TRIzol-RT-PCR method.
Significance and Impact of the Study:  This is the first study to detect NVs with IMS/RT-PCR using Mab, and could serve as a model for future assays when broadly reactive NVs-specific Mabs are developed.     

Optimising the Use of TRIzol-extracted Proteins in Surface Enhanced Laser Desorption/ Ionization (SELDI) Analysis

Background  

Research with clinical specimens is always hampered by the limited availability of relevant samples, necessitating the use of a single sample for multiple assays. TRIzol is a common reagent for RNA extraction, but DNA and protein fractions can also be used for other studies. However, little is known about using TRIzol-extracted proteins in proteomic research, partly because proteins extracted from TRIzol are very resistant to solubilization.     

A simple and loss-free method to remove TRIzol contaminations from minute RNA samples

Acid guanidium phenol preparations such as TRIzol allow the reproducible isolation of high-quality total RNA from various sources. However, if applied to minimal sample sizes, the quality parameters of the isolated RNA are often low. Here we present an approach to improve the 260/280- and 230/260-nm ratios of such preparations as well as the ratio of the 18S/28S RNA. A simple extraction with 1-butanol eliminates smearing of the 28 S RNA and restores the characteristic ultraviolet (UV) spectrum of highly purified RNA. Application of the method is demonstrated for fluorescence-activated cell sorting (FACS)-sorted cells where the population of interest is often small.