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Results for “RNA quality”

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At least 559 records · Page 31Linked to original sources
PubMed2000

Quantitative determination of a chemically modified hammerhead ribozyme in blood plasma using 96-well solid-phase extraction coupled with high-performance liquid chromatography or capillary gel electrophoresis.

Versatile bioanalytical assays to detect chemically stabilized hammerhead ribozyme and putative ribozyme metabolites from plasma are described. The extraction protocols presented are based on serial solid-phase extractions performed on a 96-well plate format and are compatible with either IEX-HPLC or CGE back-end analysis. A validation of both assays confirmed that both the HPLC and the CGE methods possess the required linearity, accuracy, and precision to accurately measure concentrations of hammerhead ribozyme extracted from plasma. These methods should be of general use to detect and quantitate ribozymes from other biological fluids such as serum and urine.

Animals↗
PubMed2000

Analysis of mutations in oral poliovirus vaccine by hybridization with generic oligonucleotide microchips.

This paper describes use of a new technology of hybridization with a micro-array of immobilized oligonucleotides for detection and quantification of neurovirulent mutants in Oral Poliovirus Vaccine (OPV). We used a micro-array consisting of three-dimensional gel-elements containing all possible hexamers (total of 4096 probes). Hybridization of fluorescently labelled viral cDNA samples with such microchips resulted in a pattern of spots that was registered and quantified by a computer-linked CCD camera, so that the sequence of the original cDNA could be deduced. The method could reliably identify single point mutations, since each of them affected fluorescence intensity of 12 micro-array elements. Micro-array hybridization of DNA mixtures with varying contents of point mutants demonstrated that the method can detect as little as 10% of revertants in a population of vaccine virus. This new technology should be useful for quality control of live viral vaccines, as well as for other applications requiring identification and quantification of point mutations.

DNA Mutational Analysis↗
PubMed1999

Quantification of murine cytokine mRNAs using real time quantitative reverse transcriptase PCR.

Recently, a novel technique for "real time" quantitative Reverse Transcriptase-PCR which measures PCR-product accumulation during the exponential phase of the PCR reaction using a dual-labelled fluorogenic probe, has been developed. This method allows direct detection of PCR-product formation by measuring the increase in fluorescent emission continuously during the PCR reaction. Here we present data validating this PCR-method for the quantification of murine cytokines and other factors playing a role in immune regulation (IL-1, IL-2, IL-4, IL-5, IL-6, IL-7, IL-10, IL-12p40, IL-13, IL-15, IFN-gammaTNF-alphaTGF-beta and iNOS). For each substance of interest, a set of primers and internal probe was designed, which specifically amplify the target cDNA, not co-amplifying contaminating genomic DNA. Furthermore, a corresponding reference plasmid cDNA clone was constructed, allowing direct quantification. Additionally, normalization to the housekeeping genes beta-actin or GAPDH was performed. The assay is very sensitive and accurate. It is a "closed-tube" PCR reaction, avoiding time-consuming and hazardous post-PCR manipulations and decreasing the potential risk of PCR contamination.

Animals↗
PubMed2001

Facts and pitfalls in the detection of tyrosinase mRNA in the blood of melanoma patients by RT-PCR.

Reverse transcription (RT) of tyrosinase mRNA and specific cDNA amplification to facilitate the early detection of circulating tumor cells in melanoma patients have been reported. The significance and practical value of these procedures for the diagnosis of tumor dissemination in melanoma patients is, however, still unclear. We analyzed peripheral blood samples of melanoma patients of different clinical stages for the presence of tyrosinase mRNA by reverse transcriptase polymerase chain reaction (RT-PCR). In addition to a nested RT-PCR-based system, we evaluated the new PCR enzyme-linked immunosorbent assay tyrosinase system for sensitivity and specificity in detecting circulating melanoma cells. Our results showed a high sensitivity and specificity for this system in detecting one melanoma cell in 1 ml of whole blood. Using different methods of detection, no tyrosinase mRNA was detectable in blood samples of patients with primary melanoma and regional lymph node metastases. In a small number of patients with visceral metastases (10-30%), we found tyrosinase mRNA-positive results. Analyses of different blood samples taken at 2-h intervals indicate that tumor cells persist only transiently in the peripheral blood. Successful establishment of melanoma cell growth from tyrosinase mRNA-positive samples indicates that viable tumor cells exist in melanoma patients' peripheral blood. Our results indicate a low amount of tyrosinase-specific transcripts in a small subset of stage IV patients and suggest that the analysis of tyrosinase mRNA in peripheral blood samples is not helpful as a prognostic marker or monitoring tool in melanoma patients.

Humans↗
PubMed1995

The relative importance of premortem acidosis and postmortem interval for human brain gene expression studies: selective mRNA vulnerability and comparison with their encoded proteins.

To help account for the variable quality and quantity of RNA in human brain, we have studied the effect of premortem (agonal state) and postmortem factors on the detection of poly(A)+mRNA and eight mRNAs. For comparison, the influence of the same factors upon gene products encoded by the mRNAs was studied immunocytochemically or by receptor autoradiography. Brain pH declined with increasing age at death and was related to agonal state severity, but was independent of postmortem interval and the histological presence of hypoxic changes. By linear regression, pH was significantly associated with the abundance of several of the RNAs, but not with poly(A)+mRNA, immunoreactivities, or binding site densities. Postmortem interval had a limited influence upon mRNA and protein products. Freezer storage time showed no effect. Parallel rat brain studies showed no relationship between postmortem interval (0-48 h) and amounts of total RNA, poly(A)+RNA, or two individual mRNAs; however, RNA content was reduced by 40% at 96 h after death. pH is superior to clinical assessments of agonal state or mode of death in predicting mRNA preservation. It provides a simple means to improve human brain gene expression studies. pH is stable after death and during freezer storage and can be measured either in cerebrospinal fluid or in homogenised tissue.

Acidosis↗
PubMed1993

Analytical procedures and quality assurance criteria for the determination of major and minor deoxynucleosides in fish tissue DNA by liquid chromatography-ultraviolet spectroscopy and liquid chromatography-thermospray mass spectrometry.

Analytical procedures and quality assurance criteria have been established for enzymatic hydrolysis of fish tissue DNA to free nucleosides and their subsequent characterization by liquid chromatography-photodiode-array ultraviolet spectroscopy and liquid chromatography-thermospray mass spectrometry. Optimization of enzymatic efficiency to assure minimal loss of modified nucleosides is described. Variability in analyte capacity factors and multiwavelength response have been compared for analyte standards and hydrolysates, and results have been used to derive qualitative and quantitative quality assurance criteria. A comparison of DNA mole percent calculated using single-wavelength quantification and multiwavelength averaging quantification indicates that less variability in data may be expected using the multiwavelength technique. Finally, the composition of DNA from liver of three species of fish widely used in mutagen/carcinogen laboratory and field studies, rainbow trout (Onchorynchus mykiss), medaka (Oryzias latipes), and brown bullhead (Ictalurus nebulosus), has been determined. Identification of deoxyuridine in the DNA hydrolysates of each fish indicates that this analyte should be measured to accurately report DNA deoxynucleoside mole percent, especially when reporting data for the methylation of deoxycytidine.

Animals↗
PubMed2004

External quality control assessment in PCR diagnostics of dengue virus infections.

BACKGROUND: Increased travelling to countries endemic for dengue fever (DF) demands efficient laboratory diagnostics. Nucleic acid amplification techniques (NAT) are now frequently used for rapid diagnosis of imported viral diseases. Different PCR systems are available. OBJECTIVES: In order to assess the quality of molecular diagnostics of dengue virus infections, an external quality assurance (EQA) in PCR diagnostics was conducted. STUDY DESIGN: A panel of 10 human plasma samples was prepared and spiked with dengue virus types DEN-1 to DEN-4. In addition, a 10-fold dilution series (1:10-1:10(4) ) of DEN-3 virus was included. The panel was pre-tested by nested RT-PCR, in-house real-time PCR, and a commercial real-time PCR kit. The samples were inactivated by gamma irradiation and shipped in freeze dried state. Thirteen laboratories, within the European network for the diagnostics of imported viral diseases (ENIVD) took part using either single-round, nested, or real-time RT-PCR methods. Two laboratories used two methods in parallel, summarising up to 15 comparable results. RESULTS: 33-100% correct results were achieved. All laboratories detected DEN-2 correctly, followed by DEN-1 (14 positive results of 15), DEN-3 (12/15) and DEN-4 (11/15). Testing of the serial dilution revealed low sensitivity in many labs, with results ranging from 33 to 80% of correctly tested samples. CONCLUSION: The EQA gives a feedback of the quality of the RT-PCR system used by each respective laboratory. The different test systems and amplification conditions demonstrate the importance of external quality control measures.

Animals↗
PubMed2005

A multi-centre pilot proficiency programme to assess the quality of molecular detection of respiratory viruses.

OBJECTIVES: To assess the quality of molecular detection of respiratory viruses in clinical diagnostic laboratories. STUDY DESIGN: Respiratory virus proficiency panels were produced from diluted stocks of respiratory viruses provided and tested by four reference laboratories. The panels consisted of strong positive, positive, low positive and negative samples for influenza viruses A and B, respiratory syncytial virus, parainfluenza viruses 1 and 3, adenovirus serotypes 4 and 7, human rhinovirus serotypes 16, 72 and 90, human coronaviruses OC43 and 229E. The panels were sent to 17 participants; results and information on methodology was collected. RESULTS: All laboratories returned results, of which five submitted complete data sets. So, for analysis all results were combined. Samples were correctly identified by participants in 93.75%, 76.75% and 47.03% for the high positive, positive and low positive samples, respectively. One false positive was reported for all data sets (1.1%). The overall score for all assays using different methodologies was 78.8%. Laboratory performance was not dependant on methodology as all in-house methodologies could achieve optimal results, but dependant on careful optimisation and procedures specific to the laboratory. CONCLUSIONS: The first proficiency panel showed that in general all participants performed well. Although, it also highlights areas for improvement for all participants in order to generate robust results for use in clinical diagnostics.

Adenoviruses, Human↗
PubMed2006

Isolation of neutrophils from mouse liver: A novel method to study effector leukocytes during inflammation.

Neutrophils are phagocytic leukocytes that represent one of the first lines of defense during infection and injury. Neutrophils emigrate into tissues during inflammation and are phenotypically different compared to cells in the circulation. To further understand the biology of tissue-recruited neutrophils, we have developed a reliable method to isolate these cells from inflamed liver. Acute liver inflammation was induced in mice by systemic treatment with adenovirus vectors. Two hours following adenovirus treatment, livers were enzymatically digested and leukocytes isolated by Percoll density gradient centrifugation. Neutrophils were then purified by negative immunomagnetic separation. Neutrophils isolated in this manner were 95% pure as determined by flow cytometry and more than 97% viable by propidium iodide staining. In order to carry out molecular studies, we extracted high quality genomic DNA and RNA from isolated neutrophils. PCR was used to successfully amplify sample genes from isolated neutrophil DNA. Isolated neutrophil RNA was used in a ribonuclease protection assay to evaluate chemokine gene expression. Neutrophils were shown to express multiple chemokine mRNA transcripts including MIP-1 beta, MIP-2 and IP-10. This work describes a novel method to isolate highly pure, viable neutrophils from pathologically inflamed tissue for subsequent detailed cellular and molecular analysis.

Adenoviridae↗
PubMed2005

Simultaneous measurement of gene expression for hepatotoxicity in thioacetamide-administered rats by DNA microarrays.

DNA microarray technology was developed as a tool for simultaneously measuring a number of gene expression changes, and has been applied for investigations of toxicity assessments of chemicals. In this study, we used a typical hepatotoxicant, thioacetamide (TA), to find correlations between the extent of hepatotoxicity and certain gene expression patterns or specific gene expression profiles. TA was intraperitoneally administered at high (400 mg/kg), medium (150 mg/kg) or low (50 mg/kg) dose (four rats per group) and then the serum and liver were collected at the indicated time (6, 12, 24, 36 and 48 h). Serum biochemical markers were measured and hepatic mRNA expression profiles were analyzed by a DNA microarray. Serum aspartate aminotransferase (AST) and alanine aminotransferase (ALT) were increased by TA-administration in a dose-dependent manner and reached the maximum at 24h. Hierarchical clustering analysis of all dosage groups revealed in 2 major clusters, distinguished by an early (6 and 12h) and a late (24, 36 and 48 h) phase. The early and late phase clusters were sorted in time- and dose-dependent manners. The major gene expression profile obtained by quality-threshold (QT) clustering analysis showed the same maximal toxic time as that estimated by the serum biochemical markers. The individual expression profiles of the candidate genes selected in our previous studies and the simultaneous gene expression patterns measured by five typical hepatotoxicants including TA also reflected the hepatotoxicity of TA. These findings suggest that the potential toxic effects appearing as gene expression changes are independent of the dosage of TA. This study suggested that the major gene expression profile estimated by QT clustering would be a sensitive marker of hepatotoxicity.

Alanine Transaminase↗
PubMed2005

The transcriptome of HCV replicon expressing cell lines in the presence of alpha interferon.

We have used DNA microarray analysis of human hepatoma and epithelial carcinoma cells expressing hepatitis C virus (HCV) subgenomic replicons to test whether HCV replication alters gene expression and influences the alpha interferon (IFN-alpha) response. We directly compared the HCV replicon system with a similar system based on a subgenomic replicon of the West Nile virus (WNV) subtype Kunjin virus. We found that in contrast to WNV replicons, persistent replication of HCV replicons did not significantly alter the transcriptome of infected cells nor did it inhibit the nature of the IFN-stimulated genes (ISGs). Our results also provided evidence for the existence of a small number of ISGs that could play a role in the inhibition of HCV replication by IFN-alpha. Finally, we identified ISGs that are activated by the cytokine in a cell-type specific fashion.

Cell Line↗
PubMed2005

Production of custom microarrays for neuroscience research.

Microarray chips produced by commercial vendors and academic laboratories are mostly generic in nature to facilitate wide applicability. With the sequencing of the human, mouse, and rat genomes, the thrust is to expand clone and oligonucleotide sets and increase the number of genes represented on a particular array. This is appropriate for discovery based investigations where microarray technology has been successfully utilized. However, array technology can also be employed to perform hypothesis based studies if optimized chips can be produced with relevant content. Existing array technology available at core facilities can be effectively utilized to produce a custom microarrays with genes that are most relevant to the research interests of individual investigators or research groups for use as a standard molecular tool. The power of this technology can be harnessed to further our understanding of specific biological problems without involvement in extensive data mining and analysis. The custom microarray approach is presented with procedural details for design and production in the context of neurobiological investigations.

Animals↗
PubMed2004

Gene-specific dye bias in microarray reference designs.

The most widely used microarray experiment design includes the use of a reference standard. Comparisons of gene expression between samples are facilitated because each sample is directly measured against the reference standard, using two fluorescent dyes. Numerous reports indicate that some genes incorporate the two commonly used dyes with different efficiencies, contributing to inaccurate data. However, it is widely assumed that these effects will not corrupt results if the reference standard is labeled with the same dye on each microarray. We demonstrate that this assumption is not reliable and that dye orientation can significantly influence measured changes in gene expression.

Animals↗
PubMed2006

Identification and validation of suitable endogenous reference genes for gene expression studies of human bladder cancer.

PURPOSE: Housekeeping genes as endogenous references are generally used for the relative quantification of target genes in gene profiling studies. To date that issue has not been sufficiently investigated in bladder cancer. From a panel of 9 potential candidates we selected the most stable housekeeping genes for gene normalization in bladder cancer tissue. MATERIALS AND METHODS: Expression profiles of the 9 genes ACTB, ALAS1, G6PD, GAPD, HMBS, HPRT1, K-ALPHA-1, SDHA and TBP were established in matched malignant and nonmalignant tissue specimens from 14 patients with bladder cancer. Quality assessment of isolated RNA was performed with a 2100 Bioanalyzer (Agilent Technologies, Palo Alto, California) and real-time reverse transcriptase-polymerase chain reaction was performed with LightCycler. The software geNorm and NormFinder (Aarhus University, Aarhus, Denmark) were used to identify the most suitable reference genes. RESULTS: RNA was isolated with high purity and integrity. Candidate reference genes showed a broad range of between 20 and 34 polymerase chain reaction cycles. Expression did not depend on patient sex or tumor stage. GAPD, G6PD and HMBS showed significant differences in expression between malignant and nonmalignant pairs (at least p <0.04). Expression of the remaining genes did not differ between the matched pairs. SDHA and TBP were the most stably expressed genes, covering higher and lower expression levels. CONCLUSIONS: For normalization purposes in gene profiling studies of bladder cancer the genes SDH and TBP are recommended as single reference genes depending on the expression level of the target gene or more favorably in combination.

Aged↗
PubMed2006

Troubleshooting microarray hybridizations.

Microarray experiments are being performed more widely than ever before, but even seasoned investigators can experience technical problems with hybridizations. This chapter provides guidelines for recognizing, rectifying, and avoiding common trouble areas. Specifically, it addresses frequent complications related to artifacts of printing, RNA sample preparation and quality, fluorophore labeling, hybridization conditions, and posthybridization washes. Emphasis is placed on investigating problems though a combination of appropriate controls and image analysis, where diagnostic plots of data quality are used to illustrate characteristics of acceptable and unsatisfactory hybridizations. This chapter also discusses resources available to microarray users hoping to improve the sensitivity and specificity of their experiments.

Oligonucleotide Array Sequence Analysis↗
PubMed2003

Differentiation between porcine epidemic diarrhea virus and transmissible gastroenteritis virus in formalin-fixed paraffin-embedded tissues by multiplex RT-nested PCR and comparison with in situ hybridization.

Porcine epidemic diarrhea virus (PEDV) and transmissible gastroenteritis virus (TGEV) were detected and differentiated in formalin-fixed, paraffin-embedded tissues from experimentally and naturally infected pigs by multiplex reverse transcription-nested polymerase chain reaction (RT-nPCR). The results of this new method were compared with in situ hybridization. A method based on xylene deparaffinization followed by proteinase K digestion yielded RNA of a suitable quality for reliable and consistent multiplex RT-nPCR analyses. PEDV and TGEV cDNAs were detected in jejunal tissues from experimentally and naturally infected pigs by multiplex RT-nPCR. Distinct positive signals for PEDV and TGEV were also detected in the same jejunal tissues by in situ hybridization. The rate of conformity between multiplex RT-nPCR and in situ hybridization was 100% for the detection of PEDV and TGEV in formalin-fixed paraffin-embedded jejunal tissues.

Animals↗
PubMed2000

Delimination of brewing yeast strains using different molecular techniques.

In general, the genetic characteristics, the phenotype and the microbial purity of the production brewing yeast strains are among the most important factors in maintaining a consistently good quality of products. Analysis of restriction fragment length polymorphism (RFLP) patterns of 18S rRNA-coding DNA was investigated to group ale and lager strains. All production brewing yeast strains showed the same RFLP pattern as the type strain and synonym type strains of S. cerevisiae, and were quite different from the type and synonym type strains of S. pastorianus. Based on these data, all production brewing yeast strains investigated in this study appeared to belong to S. cerevisiae. Electrophoretic karyotyping and random amplified polymorphic DNA (RAPD) analysis appeared to be suitable methods for distinguishing not only the type and synonym type strain of S. cerevisiae and S. pastorianus, but also the ale and the lager strains.

Beer↗