应用循环逆转录PCR技术检测丙型肝炎病毒RNA

循环逆转录（ｃｉｒｃｕｌａｔｏｒｙ ｒｅｖｅｒｓｅ ｔｒａｎｓｃｒｉｐｔｉｏｎ,ＣＲＴ）是线性增长逆转录ｃＤＮＡ产量的一种新技术。为了将该技术用于检测ＨＣＶ ＲＮＡ,通过改变ＣＲＴ的循环次数,结合竞争ＰＣＲ,作出标准曲线。采用１６次ＣＲＴ加３４次循环ＰＣＲ检测了１３６例ＨＣＶ ＥＬＩＳＡ阳性、５４例ＨＣＶ ＥＬＩＳＡ阴性和１０８例临床可疑病人全血标本,并与逆转录ＰＣＲ（ＲＴ－ＰＣＲ）和巢式ＰＣＲ（     

Isolation of High Quality RNA from Oilseeds of Jatropha curcas

High quality RNA with good yield is a prerequisite for carrying out several molecular biology studies. Recalcitrant tissues such as oilseeds pose several problems while isolating good quality RNA. We have standardized a fast and simple protocol for RNA isolation from the seeds of Jatropha curcas, which gives good quality RNA without compromising for the yield. By including pre wash of seed powder with acetone and removal of polysaccharides through selective precipitation, we have been regularly isolating good quality total RNA in the range of 300–450 μg g^?1 depending upon tissue type. The RNA isolated by this procedure is devoid of any contaminating DNA. The RNA preparations have been subjected to cDNA synthesis and PCR, and found suitable for these studies. This method also works satisfactorily with groundnut and mustard seeds.     

小麦总RNA的快速提取

Rneasy Kits用于从小量小麦中分离总RNA。它为同时和多次制备各种生物样品提供了一种快捷而简便的方法-整个过程可以在30分钟内完成。而且Rneasy方法中不再涉及使用有毒物质,如酚类、氯仿等。通过此方法纯化的RNA可用于各种标准的下游技术,如RT-PCR、polyA^ RNA选择、差异显示技术、Northern点杂交和狭线杂交、引物延伸、cDNA合成、陈列表达分析和表达芯片分析等。使用这个盒子,我们多次成功的进行了小麦总RNA的分离,其中的三次在本文中进行了分析。OD260nm/OD280nm介于1.7-2.0之间,OD260nm/OD230nm大于2.0.说明RNA纯度很高,未被蛋白质、苯酚等物质污染。     

RNA folding using quantum computers

The 3-dimensional fold of an RNA molecule is largely determined by patterns of intramolecular hydrogen bonds between bases. Predicting the base pairing network from the sequence, also referred to as RNA secondary structure prediction or RNA folding, is a nondeterministic polynomial-time (NP)-complete computational problem. The structure of the molecule is strongly predictive of its functions and biochemical properties, and therefore the ability to accurately predict the structure is a crucial tool for biochemists. Many methods have been proposed to efficiently sample possible secondary structure patterns. Classic approaches employ dynamic programming, and recent studies have explored approaches inspired by evolutionary and machine learning algorithms. This work demonstrates leveraging quantum computing hardware to predict the secondary structure of RNA. A Hamiltonian written in the form of a Binary Quadratic Model (BQM) is derived to drive the system toward maximizing the number of consecutive base pairs while jointly maximizing the average length of the stems. A Quantum Annealer (QA) is compared to a Replica Exchange Monte Carlo (REMC) algorithm programmed with the same objective function, with the QA being shown to be highly competitive at rapidly identifying low energy solutions. The method proposed in this study was compared to three algorithms from literature and, despite its simplicity, was found to be competitive on a test set containing known structures with pseudoknots.     

RNA extraction from human articular cartilage by chondrocyte isolation

We report an optimized method for RNA extraction from human articular cartilage that does not require the use of specialized equipment or column purification. To maximize RNA yield while minimizing degradation and contamination, chondrocytes are isolated from the extracellular matrix and the traditional TRIzol protocol is modified to include two RNA-DNA-protein phase separations. We compared RNA extracted using this modified method with the traditional TRIzol method by spectrophotometry, Bioanalyzer, and real-time polymerase chain reaction (PCR). With the modified method, RNA recovery is increased by nearly 1μg per 100mg of cartilage, and RNA integrity number (RIN) is improved from 2.0 to 7.5.     

A ribonuclease protection assay for the direct detection and quantitation of hepatitis C virus RNA

A ribonuclease protection assay (RPA) was developed for the direct detection and quantitation of HCV RNA in infected patients' sera or plasma using HCV [(32)P]RNA from the conserved 5'-untranslated region (5'-UTR) as a probe. We were able to directly detect the presence of HCV RNA by RPA in several infected patients' samples. The viremic status of HCV infected patients with indeterminate recombinant immunoblot assay (RIBA II) was also determined by this assay. Polymerase chain reaction (PCR) was also performed on all these samples and were found to be positive with a concordance of 100% between the results of PCR and RPA. The RPA was able to detect approximately 1 pg of HCV RNA. A limited sequence heterogeneity among HCV isolates was also observed by this assay, suggesting that this may be a faster method of detecting heterogeneous HCV sequences in patients' samples. This simple and specific method could be used to quantitate HCV RNA in order to better determine viremia and follow the course of HCV infection especially when RIBA II results are indeterminate.     

High‐throughput quantification of chloroplast RNA editing extent using multiplex RT‐PCR mass spectrometry

RNA editing in plants, animals, and humans modifies genomically encoded cytidine or adenosine nucleotides to uridine or inosine, respectively, in mRNAs. We customized the MassARRAY System (Sequenom Inc., San Diego, CA, USA, www.sequenom.com ) to assay multiplex PCR‐amplified single‐stranded cDNAs and easily analyse and display the captured data. By using appropriate oligonucleotide probes, the method can be tailored to any organism and gene where RNA editing occurs. Editing extent of up to 40 different nucleotides in each of either 94 or 382 different samples (3760 or 15 280 editing targets, respectively) can be examined by assaying a single plate and by performing one repetition. We have established this mass spectrometric method as a dependable, cost‐effective and time‐saving technique to examine the RNA editing efficiency at 37 Arabidopsis thaliana chloroplast editing sites at a high level of multiplexing. The high‐throughput editing assay, named Multiplex RT‐PCR Mass Spectrometry (MRMS), is ideal for large‐scale experiments such as identifying population variation, examining tissue‐specific changes in editing extent, or screening a mutant or transgenic collection. Moreover, the required amount of starting material is so low that RNA from fewer than 50 cells can be examined without amplification. We demonstrate the use of the method to identify natural variation in editing extent of chloroplast C targets in a collection of Arabidopsis accessions.     

Poliovirus RNA recombination: mechanistic studies in the absence of selection.

Direct and quantitative detection of recombinant RNA molecules by polymerase chain reaction (PCR) provides a novel method for studying recombination in RNA viruses without selection for viable progeny. The parental poliovirus strains used in this study contained polymorphic marker loci approximately 600 bases apart; both exhibited wild-type growth characteristics. We established conditions under which the amount of PCR product was linearly proportional to the amount of input template, and the reproducibility was high. Recombinant progeny were predominantly homologous and arose at frequencies up to 2 x 10(-3). Recombination events increased in frequency throughout replication, indicating that there is no viral RNA sequestration or inhibition of recombination late in infection as proposed in earlier genetic studies. Previous studies have demonstrated that poliovirus recombination occurs by a copy-choice mechanism in which the viral polymerase switches templates during negative-strand synthesis. Varying the relative amount of input parental virus markedly altered reciprocal recombination frequencies. This, in conjunction with the kinetics data, indicated that acceptor template concentration is a determinant of template switching frequency. Since positive strands greatly outnumber negative strands throughout poliovirus infection, this would explain the bias toward recombination during negative-strand synthesis.     

Structural architecture of an RNA that competitively inhibits RNase L

Activation of RNase L endonuclease activity is part of the mammalian innate immune response to viral infection. The poliovirus RNA genome contains a sequence in its protein-coding region that can act as a competitive inhibitor of RNase L. Mutation, sequence, and functional analysis of this competitive inhibitor RNA (ciRNA) revealed that its activity depends on specific sequences, showed that a loop-loop hairpin interaction forms in the ciRNA, and suggested the presence of a loop E motif. These features lead to the hypothesis that the ciRNA's function is conferred in part by a specific three-dimensional folded RNA architecture. By using a combination of biophysical, mutational, and functional studies, we have mapped features of the three-dimensional architecture of the ciRNA in its unbound form. We show that the loop-loop interaction forms in the free ciRNA and affects the overall structure, perhaps forming long-range tertiary interactions with the loop E motif. Local tight RNA-RNA backbone packing occurs in parts of the structure, but the fold appears to be less stable than many other tightly packed RNAs. This feature may allow the ciRNA to accommodate the translocation of ribosomes and polymerase across this multifunctional region of the viral RNA but also to function as an RNase L inhibitor.