一种用矽石快速提取总RNA的方法

根据矽石能与核酸结合的特性,建立了用矽石提取细胞和组织总RNA的方法,方法简便、快速,所得RNA适用于各种研究。     

A procedure for the small-scale isolation of plant RNA suitable for RNA blot analysis

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.     

迟钝爱德华氏菌RNA提取及痕量基因组DNA去除方法探究

迟钝爱德华氏菌EIB202是一类细胞壁结构特殊的革兰氏阴性菌,高质量RNA提取相对较难。为了从转录组水平研究这类致病菌的致病机理,需要摸索有效的RNA提取及RNA样品中痕量基因组DNA去除方法。对常规RNA提取步骤进行改进,增加PBS清洗、反复冻融及较高浓度溶菌酶处理等步骤;另外,利用小体系基因组DNA去除系统,Mg2+与Mn2+协同激活DNase I去除RNA样品中基因组DNA污染。利用优化方法提取的RNA在质量及浓度(1 740 ng/μL)方面均有了显著改善,并建立了一套完全去除RNA样品中痕量基因组DNA污染的程序。     

High-quality RNA from cells isolated by laser capture microdissection

Laser capture microdissection (LCM) provides a rapid and simple method for procuring homogeneous populations of cells. However, reproducible isolation of intact RNAfrom these cells can be problematic; the sample may deteriorate before or during sectioning, RNA may degrade during slide staining and LCM, and inadequate extraction and isolation methods may lead to poor recovery. Our report describes an optimized protocol for preparation of frozen sections for LCM using the HistoGene Frozen Section Staining Kit. This slide preparation method is combined with the PicoPure RNA Isolation Kitfor extraction and isolation of RNA from low numbers of microdissected cells. The procedure is easy to perform, rapid, and reproducible. Our results show that the RNA isolated from the LCM samples prepared according to our protocol is of high quality. The RNA maintains its integrity as shown by RT-PCR detection of genes of different abundance levels and by electrophoretic analysis of ribosomal RNA. RNA obtained by this method has also been used to synthesize probes for interrogating cDNA microarray analyses to study expression levels of thousands of genes from LCM samples.     

Transfection of RNA from Organ Samples of Infected Animals Represents a Highly Sensitive Method for Virus Detection and Recovery of Classical Swine Fever Virus

Translation and replication of positive stranded RNA viruses are directly initiated in the cellular cytoplasm after uncoating of the viral genome. Accordingly, infectious virus can be generated by transfection of RNA genomes into susceptible cells. In the present study, efficiency of conventional virus isolation after inoculation of cells with infectious sample material was compared to virus recovery after transfection of total RNA derived from organ samples of pigs infected with Classical swine fever virus (CSFV). Compared to the conventional method of virus isolation applied in three different porcine cell lines used in routine diagnosis of CSF, RNA transfection showed a similar efficiency for virus rescue. For two samples, recovery of infectious virus was only possible by RNA transfection, but not by the classical approach of virus isolation. Therefore, RNA transfection represents a valuable alternative to conventional virus isolation in particular when virus isolation is not possible, sample material is not suitable for virus isolation or when infectious material is not available. To estimate the potential risk of RNA prepared from sample material for infection of pigs, five domestic pigs were oronasally inoculated with RNA that was tested positive for virus rescue after RNA transfection. This exposure did not result in viral infection or clinical disease of the animals. In consequence, shipment of CSFV RNA can be regarded as a safe alternative to transportation of infectious virus and thereby facilitates the exchange of virus isolates among authorized laboratories with appropriate containment facilities.     

Total RNA isolation from recalcitrant yeast cells

Conventional methods of RNA isolation are not suitable for yeast cells from stationary phase and fermentation broth. Methods specially reported for such cells are cumbersome and do not lend themselves for use with large number of samples. Here we report a facile method of RNA isolation from such recalcitrant yeast cells. The entire procedure is performed in microcentrifuge tubes and, thus, is ideal for faster processing of multiple samples. The method consistently gives high quality and quantity of RNA, which was found to be suitable for downstream applications such as quantitative real-time polymerase chain reaction. Besides Saccharomyces cerevisiae, the method was found to work equally well with other yeast species; thus, it is likely to have wider applicability.     

Improved method for the isolation of RNA from plant tissues

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.     

从血液中提取总RNA的一种快速高效方法

血液中含有大量的RNA酶 ,可引起RNA的降解 .防止RNA酶的降解 ,是保证所得RNA片段完整的关键 .目前提取RNA的方法较多 ,但有些方法尚不能完全防止RNA降解 .将TRIZOL方法稍加改进 ,将TRIZOL与异硫氰酸胍联用提取血液淋巴细胞总RNA .琼脂糖凝胶电泳结果表明 ,其 2 8SRNA与 18SRNA的比值为 2∶1,优于单独使用其中任何一种试剂者 .此方法同样适用于从其它细胞中提取RNA .     

CKC: Isolation of Nucleic Acids from a Diversity of Plants Using CTAB and Silica Columns

To assay for viruses in plant samples, we required a method for nucleic acid isolation that is rapid, simple, and applicable to the widest possible variety of plants. A protocol for isolation of total nucleic acid (TNA) was developed by combining common CTAB methods with silica spin columns. We report data on TNA purity and RNA quality from over 30 plant genera representing 25 families. Measurements showed that RNA is of high quality, and one-step RT-PCR was successfully performed on all samples. The protocol can be completed in less than 2 h.     

A micromethod for the isolation of total RNA from adipose tissue.

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.     

ISOLATION OF HIGH-QUALITY RNA FROM GRAINS OF DIFFERENT MAIZE VARIETIES

The study of gene expression in maize varieties represents a powerful tool aiming to increase vitamin A precursors. However, the isolation of RNA from different maize varieties is challenging because these varieties show different levels of polysaccharides, and most methods available for RNA isolation are inappropriate for grain samples. The polysaccharides co-purify and co-precipitate with RNA during isolation, resulting in low-quality RNA, compromising the use of RNA in subsequent applications. Thus, a cetyltrimethylammonium bromide (CTAB)-based method was adapted in this study and compared with six methods for RNA isolation, including commercial reagents and RNA and DNA isolation kits, in order to identify the most appropriate for maize grains from different varieties. Most of the methods evaluated were considered inadequate due to limitations in terms of purity and/or quantity of the isolated RNA, which affected the efficiency of subsequent RT-qPCR analysis, resulting in nonamplification of β-carotene hydroxylase gene (HYD3) or high deviation among replicates. However, the CTAB modified method allowed the study to obtain intact RNA, with high quality and quantity, from 25 maize varieties. Furthermore, this RNA was successfully used to evaluate the expression of HYD3 gene by real-time qualitative polymerase chain reaction (RT-qPCR), and thus represents a simple, efficient, and low-cost strategy.     

Direct Isolation of High-Quality Low Molecular Weight RNA of Pear Peel from the Extraction Mixture Containing Nucleic Acid

Low molecular weight RNA (LMW RNA) is generally obtained either from the total RNA or from total nucleic acids solution. Many steps and chemical reagents are involved in traditional methods for LMW RNA isolation where degradation of LMW RNA often occurs, especially for plant materials with high levels of secondary catabolites. In this study, an efficient method was developed to directly isolate pure LMW RNA from pear peel, a material rich in polyphenolics that is covered with a layer of wax. The method was based on polyethylene glycol (PEG) precipitation combining CTAB buffer which is often used to isolate RNA from polysaccharide-rich and polyphenolics-rich materials. The entire procedure could be completed within 6 h and many samples could be processed at the same time. Few and common chemicals are used with this method. Hence, it could be used as an ordinary method in the laboratory. The developed method was further tested by isolating LMW RNA from Arabidopsis. Using the isolated LMW RNA samples, microRNAs were successfully detected and characterized.     

Isolation of RNA from bacterial samples of the human gastrointestinal tract

The human gastrointestinal (GI) tract contains a complex microbial community that consists of numerous uncultured microbes. Therefore, nucleic-acid-based approaches have been introduced to study microbial diversity and activity, and these depend on the proper isolation of DNA, rRNA and mRNA. Here, we present an RNA isolation protocol that is suitable for a wide variety of GI tract samples. The procedure for isolating DNA from GI tract samples is described in another Nature Protocols article. One of the benefits of our RNA isolation protocol is that sampling can be performed outside the laboratory, which offers possibilities for implementation in large intervention studies. The RNA isolation is based on mechanical disruption, followed by isolation of nucleic acids using phenol:chloroform:isoamylalcohol extraction and removal of DNA. In our laboratory, this protocol has resulted in the isolation of rRNA and mRNA of sufficient quality and quantity for microbial diversity and activity studies. Depending on the number of samples, the sample type and the quenching procedure chosen, the whole procedure can be performed within 2.5-4 h.     

Isolation of High-Quality RNA from <Emphasis Type="Italic">Reaumuria soongorica</Emphasis>, a Desert Plant Rich in Secondary Metabolites

RNA isolation is a prerequisite for the study of the molecular mechanisms of stress tolerance in the desert plant Reaumuria soongorica, an extreme xeric semi-shrub. However, R. soongorica that contains high levels of secondary metabolites that co-precipitate with RNA, making RNA isolation difficult. Here the authors propose a new protocol suitable for isolating high-quality RNA from the leaves of R. soongorica. Based on a CTAB method described by Liu et al., the protocol has been improved as follows: the samples were ground with PVPP to effectively inhibit the oxidation of phenolics, contaminating DNA was removed with DNase I, and NaAc was used along with ethanol for precipitation to enhance the RNA yield and shorten the precipitation time. Gel electrophoresis and spectrophotometric analysis indicated that this isolation method provides RNA with no DNA contamination. Moreover, the yield (183.79 ± 40.36 μg/g) and quality were superior to those using the method of Liu et al., which yields RNA with significant DNA contamination at 126.30 ± 29.43 μg/g. Gene amplification showed that the RNA obtained using this protocol is suitable for use in downstream molecular procedures. This method was found to work equally well for isolating RNA from other desert plants. Thus, it is likely to be widely applicable.     

Identification of extracellular miRNA in human cerebrospinal fluid by next-generation sequencing

There has been a growing interest in using next-generation sequencing (NGS) to profile extracellular small RNAs from the blood and cerebrospinal fluid (CSF) of patients with neurological diseases, CNS tumors, or traumatic brain injury for biomarker discovery. Small sample volumes and samples with low RNA abundance create challenges for downstream small RNA sequencing assays. Plasma, serum, and CSF contain low amounts of total RNA, of which small RNAs make up a fraction. The purpose of this study was to maximize RNA isolation from RNA-limited samples and apply these methods to profile the miRNA in human CSF by small RNA deep sequencing. We systematically tested RNA isolation efficiency using ten commercially available kits and compared their performance on human plasma samples. We used RiboGreen to quantify total RNA yield and custom TaqMan assays to determine the efficiency of small RNA isolation for each of the kits. We significantly increased the recovery of small RNA by repeating the aqueous extraction during the phenol-chloroform purification in the top performing kits. We subsequently used the methods with the highest small RNA yield to purify RNA from CSF and serum samples from the same individual. We then prepared small RNA sequencing libraries using Illumina’s TruSeq sample preparation kit and sequenced the samples on the HiSeq 2000. Not surprisingly, we found that the miRNA expression profile of CSF is substantially different from that of serum. To our knowledge, this is the first time that the small RNA fraction from CSF has been profiled using next-generation sequencing.