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At least 1,009 records · Page 56Linked to original sources
PubMed2002

High-density oligonucleotide array analysis of mRNA transcripts in peripheral blood cells of severe atopic dermatitis patients.

BACKGROUND: There are few laboratory tests for evaluating atopic dermatitis (AD) with the exception of IgE levels or the eosinophil count. We attempted to identify new diagnostic markers by screening the genome-wide expression of transcripts in peripheral blood mononuclear cells (PBMC). METHODS: For this study, we enrolled 7 nonatopic healthy volunteers, 5 AD patients who responded well to treatment and 6 who responded poorly. We compared genome-wide transcript levels in PBMC derived from patients with severe AD and healthy volunteers using high-density oligonucleotide arrays (GeneChip, Affymetrix). After the first screening with GeneChip, we employed real-time quantitative PCR to confirm differential expression levels. RESULTS: Screening with GeneChip showed that the levels of a total of 92 transcripts increased at least 3-fold in one population compared to another. After further evaluation of these genes with real-time quantitative PCR, the levels of 4 transcripts were confirmed to be significantly different in PBMC from AD patients compared to controls, namely IFN-gamma, TRAIL (TNF-related apoptosis-inducing ligand), ISGF-3 (STAT1) and defensin-1. With the exception of IFN-gamma, none of these genes has previously been implicated in AD pathology. CONCLUSION: These 4 transcripts in PBMC are expected to be useful markers for evaluating AD.

Cells, Cultured↗
PubMed2005

Searching QTL by gene expression: analysis of diabesity.

BACKGROUND: Recent developments in sequence databases provide the opportunity to relate the expression pattern of genes to their genomic position, thus creating a transcriptome map. Quantitative trait loci (QTL) are phenotypically-defined chromosomal regions that contribute to allelically variant biological traits, and by overlaying QTL on the transcriptome, the search for candidate genes becomes extremely focused. RESULTS: We used our novel data mining tool, ExQuest, to select genes within known diabesity QTL showing enriched expression in primary diabesity affected tissues. We then quantified transcripts in adipose, pancreas, and liver tissue from Tally Ho mice, a multigenic model for Type II diabetes (T2D), and from diabesity-resistant C57BL/6J controls. Analysis of the resulting quantitative PCR data using the Global Pattern Recognition analytical algorithm identified a number of genes whose expression is altered, and thus are novel candidates for diabesity QTL and/or pathways associated with diabesity. CONCLUSION: Transcription-based data mining of genes in QTL-limited intervals followed by efficient quantitative PCR methods is an effective strategy for identifying genes that may contribute to complex pathophysiological processes.

Algorithms↗
PubMed2003

Kinetic study of porcine GM-CSF expression in porcine alveolar macrophages and spleen cells.

Granulocyte-macrophage colony-stimulating factor (GM-CSF) is an important regulator in inducing differentiation and proliferation of immune cells. The functional roles of porcine GM-CSF (pGM-CSF) have not yet been revealed. Therefore, expression patterns of pGM-CSF were investigated in immune cells after cloning and sequencing of whole pGM-CSF cDNA. Whole cDNA of pGM-CSF was amplified from porcine alveolar macrophages stimulated by lipopolysaccharide (LPS), using 5'- and 3'-rapid amplification of cDNA ends-polymerase chain reaction (RACE-PCR) methods. The products of 5'- and 3'-RACE-PCR were cloned, and the nucleotide sequence of whole pGM-CSF cDNA was determined (GenBank accession number AY116504). The kinetics of pGM-CSF mRNA expression were studied in porcine immune cells such as alveolar macrophages and spleen cells, using a real-time quantitative PCR. The expression of pGM-CSF in LPS-, phytohemagglutinin (PHA)-, or concanavalin A (ConA)-stimulated cells was always higher as compared to the control cells. The expression levels of pGM-CSF in alveolar macrophages were highest at 5 h after LPS stimulation and then continuously decreased in the late phase. In spleen cells, the LPS-stimulated group showed the highest levels after 5 h, but the PHA- and the ConA-stimulated groups showed slightly increased expression levels at the early phase and peaked at 24 h. To our knowledge, this is the first published report describing the nucleotide sequence of whole cDNA and the expression pattern of pGM-CSF using real-time quantitative PCR. These results indicate that pGM-CSF has its own characteristic expression profile in different immune cells.

Amino Acid Sequence↗
PubMed2004

Spatial and temporal distribution of two diazotrophic bacteria in the Chesapeake Bay.

The aim of this study was to initiate autecological studies on uncultivated natural populations of diazotrophic bacteria by examining the distribution of specific diazotrophs in the Chesapeake Bay. By use of quantitative PCR, the abundance of two nifH sequences (907h22 and 912h4) was quantified in water samples collected along a transect from the head to the mouth of the Chesapeake Bay during cruises in April and October 2001 and 2002. Standard curves for the quantitative PCR assays demonstrated that the relationship between gene copies and cycle threshold was linear and highly reproducible from 1 to 10(7) gene copies. The maximum number of 907h22 gene copies detected was approximately 140 ml(-1) and the maximum number of 912h4 gene copies detected was approximately 340 ml(-1). Sequence 912h4 was most abundant at the mouth of the Chesapeake Bay, and in general, its abundance increased with increasing salinity, with the highest abundances observed in April 2002. Overall, the 907h22 phylotype was most abundant at the mid-bay station. Additionally, 907h22 was most abundant in the April samples from the mid-bay and mouth of the Chesapeake Bay. Despite the fact that the Chesapeake Bay is rarely nitrogen limited, our results show that individual nitrogen-fixing bacteria have distinct nonrandom spatial and seasonal distributions in the Chesapeake Bay and are either distributed by specific physical processes or adapted to different environmental niches.

Bacteria↗
PubMed2003

Genus- and species-specific PCR primers for the detection and identification of bifidobacteria.

16SrDNA-targeted genus- and species-specific PCR primers have been developed and used for the identification and detection of bifidobacteria. These primers cover all of the described species that inhabit the human gut, or occur in dairy products. Identification of cultured bifidobacteria using PCR primer pairs is rapid and accurate, being based on nucleic acid sequences. Detection of bifidobacteria can be achieved using DNA extracted from human faeces as template in PCR reactions. We have found that, in adult faeces, the Bifidobacterium catenulatum group was the most commonly detected species, followed by Bifidobacterium longum, Bifidobacterium adolescentis, and Bifidobacterium bifidum. In breastfed infants, Bifidobacterium breve was the most frequently detected species, followed by Bifidobacterium infantis, B. longum and B. bifidum. It was notable that the B. catenulatum group was detected with the highest frequency in adults, although it has often been reported that B. adolescentis is the most common species. Real-time, quantitative PCR using primers targeting 16S rDNA shows promise in the enumeration of bifidobacteria in faecal samples. The approach to detect the target bacteria with quantitative PCR described in this review will contribute to future studies of the composition and dynamics of the intestinal microflora.

Adult↗
PubMed2000

DNA methylation and the mechanisms of CDKN2A inactivation in transitional cell carcinoma of the urinary bladder.

Alterations of the CDKN2A locus on chromosome 9p21 encoding the p16INK4A cell cycle regulator and the p14ARF1 p53 activator proteins are frequently found in bladder cancer. Here, we present an analysis of 86 transitional cell carcinomas (TCC) to elucidate the mechanisms responsible for inactivation of this locus. Multiplex quantitative PCR analysis for five microsatellites around the locus showed that 34 tumors (39%) had loss of heterozygosity (LOH) generally encompassing the entire region. Of these, 17 tumors (20%) carried homozygous deletions of at least one CDKN2A exon and of flanking microsatellites, as detected by quantitative PCR. Analysis by restriction enzyme PCR and methylation-specific PCR showed that only three specimens, each with LOH across 9p21, had bona fide hypermethylation of the CDKN2A exon 1alpha CpG-island in the remaining allele. Like most other specimens, these three specimens displayed substantial genome-wide hypomethylation of DNA as reflected in the methylation status of LINE L1 sequences. The extent of DNA hypomethylation was significantly more pronounced in TCC with LOH and/or homozygous deletions at 9p21 than in those without (26% and 28%, respectively, on average, versus 11%, p < 0.0015). No association of LOH or homozygous deletions at 9p21 with tumor stage or grade was found. The data indicate that DNA hypermethylation may be rare in TCC and that deletions are the most important mechanism for inactivation of the CDKN2A locus. The predominance of allelic loss may be explained by its correlation with genome-wide DNA hypomethylation, which is thought to favor chromosomal instability and illegitimate recombination.

Adult↗
PubMed2002

Measurement of dorsal root ganglion P2X mRNA by SYBR Green fluorescence.

The P2X receptor is a receptor-gated cationic channel that responds to ATP. The quantification of P2X mRNA expression in dorsal root ganglion (DRG) provides important information for neuropathic pain studies. We developed a rapid and sensitive external-standard-based real-time quantitative PCR assay for the quantification of mRNA of P2X receptors in mouse tissue samples. The assay uses a double-stranded DNA fluorescent dye, SYBR Green I, to continuously monitor product formation with a GeneAmp 5700 Sequence Detection System (PE Applied Biosystems). To establish the quantitative PCR amplification in a wide range of target transcripts, optimum parameters of primer sequences, concentrations of primers and/or templates, and PCR thermal protocols were experimentally determined. We also tested the reliability of this method in established experimental murine models, which were made by ligation or cutting down of the sciatic nerve. The parameters defined in this assay should be applicable to the quantification of other types of pain models and other tissue samples of mouse.

Animals↗
PubMed2006

Different expression of lipocalin-type prostaglandin D synthase in rat epididymidis.

This study was designed to explore the different expression of L-PGDS (lipocalin-type prostaglandin D synthase) in rat epididymidis and to gain further insight into the potential function of L-PGDS in male reproduction. The expression of L-PGDS in rat epididymidis was assessed using real-time quantitative PCR and immunoblotting. The distribution of L-PGDS in rat epididymidis was explored by immunohistochemical methods. The result of immunohistochemistry displayed that L-PGDS was mainly distributed in epididymidis and localized within the cytoplasm and the cilia of the epithelial cells. Real-time quantitative PCR and immunoblotting showed that L-PGDS was strikingly expressed in the caput epididymidis, while a moderate to weak expression was observed in the corpus and cauda epididymidis, the level of mRNA was 0.52+/-0.02 in the caput, 0.48+/-0.03 in the corpus and 0.32+/-0.01 in the cauda epididymidis, the level of protein expression in caput, corpus and the cauda groups was 1, 0.89+/-0.03 and 0.62+/-0.01, which suggested that L-PGDS may play certain kind of role during the process of the spermatozoa maturation.

Animals↗
PubMed2005

DNA stabilization is critical for maximizing performance of fecal DNA-based colorectal cancer tests.

We have developed a multitarget, fecal DNA screening assay that detects the presence of gene-specific mutations and long DNA fragments associated with colorectal cancer (CRC). We continue to investigate methods that may be used to optimize clinical sensitivity. The goals of this investigation are to establish how sample handling conditions affect the stability of DNA in stool, thereby potentially limiting clinical sensitivity, and to determine conditions to ameliorate DNA degradation. A study was run comparing paired sample aliquots. Quantitative PCR data for matched aliquots was used to determine first the effect of sample incubation on total recovery and integrity of DNA, then the effect of stabilization buffer addition to stool on recoverable DNA, and finally the impact of buffer addition on assay sensitivity. Comparison of quantitative PCR data for paired aliquots shows that the amount of recoverable human DNA is negatively affected by storing stool samples (N = 43) at room temperature for > or =36 hours (P = 0.0018). However, the addition of stabilization buffer leads to a significant increase in recovery of DNA (P = 0.010), compared with samples incubated without buffer. Whereas the DNA Integrity Assay (DIA) is found to be sensitive to DNA degradation (sensitivity was reduced by 82%; P = 0.0002), point mutation marker sensitivity is more refractory. Overall, DNA can be stabilized by addition of buffer to the sample, leading to increased assay sensitivity.

Buffers↗
PubMed2003

Strain variation in an emerging iridovirus of warm-water fishes.

Although iridoviruses vary widely within and among genera with respect to their host range and virulence, variation within iridovirus species has been less extensively characterized. This study explores the nature and extent of intraspecific variation within an emerging iridovirus of North American warm-water fishes, largemouth bass virus (LMBV). Three LMBV isolates recovered from three distinct sources differed genetically and phenotypically. Genetically, the isolates differed in the banding patterns generated from amplified fragment length polymorphism analysis but not in their DNA sequences at two loci of different degrees of evolutionary stability. In vitro, the isolates replicated at identical rates in cell culture, as determined by real-time quantitative PCR of viral particles released into suspension. In vivo, the isolates varied over fivefold in virulence, as measured by the rate at which they induced mortality in juvenile largemouth bass. This variation was reflected in the viral loads of exposed fish, measured using real-time quantitative PCR; the most virulent viral strain also replicated to the highest level in fish. Together, these results justify the designation of these isolates as different strains of LMBV. Strain variation in iridoviruses could help explain why animal populations naturally infected with iridovirus pathogens vary so extensively in their clinical responses to infection. The results of this study are especially relevant to emerging iridoviruses of aquaculture systems and wildlife.

Animals↗
PubMed2006

Characterization of low-acetic-acid-producing yeast isolated from 2-deoxyglucose-resistant mutants and its application to high-gravity brewing.

We isolated a mutant with low acetic acid and high ethanol productivities from 2-deoxyglucose-resistant mutants of brewers' yeast NCYC1245 (Saccharomyces cerevisiae). To determine the mechanism for these properties in the mutant (2DGR19) during fermentation, gene expression and enzyme activity related to acetic acid and ethanol production were investigated. DNA microarray analysis revealed that the transcriptional levels of many genes involved in glycolysis were higher in 2DGR19 than in NCYC1245. Among these transcriptional levels of 2DGR19 relative to NCYC1245, the expression level of ADH4 encoding alcohol dehydrogenase (ADH) was highest, which corresponded to the high ADH activity in 2DGR19. Quantitative PCR analysis also revealed that the transcriptional level of ADH4 was the highest among ADH1 to ADH4. Although no significant differences in the transcriptional levels of ALD2 to ALD6 encoding acetaldehyde dehydrogenase (ALD) between 2DGR19 and NCYC1245 were observed, ALD activity in 2DGR19 was lower. Using quantitative PCR analysis, ALD6 was found to be the most highly expressed among the ALD2 to ALD6 genes. These results indicate that ALD6 contributes to a low ALD activity, depending on post-transcriptional regulation. A high ADH activity appeared to be the major reason for the high ethanol productivity of 2DGR19. A low ALD activity was considered to be principally responsible for a low acetic acid productivity, although a high ADH activity also might have played a role. Beer brewed using 2DGR19 in pilot-scale high-gravity brewing contained about half as much acetic acid and 1.1% more ethanol compared with that brewed using NCYC1245. The use of 2DGR19 may overcome difficulties associated with high-gravity brewing.

Acetic Acid↗
PubMed2004

Real-time quantitative PCR-based serum neutralization test for detection and titration of neutralizing antibodies to chicken anemia virus.

Detection and titration of chicken anemia virus (CAV)-neutralizing antibodies has relied on tedious, time-consuming passaging of infected cells, or subjective recognition of cytopathic effect in individual cells, because CAV replicates in culture only in lymphoblastoid cell lines, and thus generates no plaques. This paper describes a rapid method, in which CAV genomes in infected cells are quantitated by qPCR 3-4 days postinfection (p.i.), without passaging cells. Three sera, weakly positive with a commercial CAV ELISA kit, from broiler chickens immunized with a commercial CAV vaccine, were used to develop the assay. Virus neutralization titers of these sera were determined using two different CAV-susceptible cell lines (MDCC-MSB1 and MDCC-CU147) by the conventional method of passaging cells infected with 10,000 TCID(50) CAV per well, and by qPCR-based methods using cells infected with 100 or 10,000 TCID(50) per well in 24-well or 96-well plates. The method was also adapted to conventional PCR. The positive sera exhibited virus neutralization activity at dilutions ranging from 1:10 to 1:320 by the various assays. Although virus neutralization titers differed somewhat depending on the assay conditions used, the relative order of the titers of the three positive sera was the same for all assays. The qPCR-based assays are as sensitive and more rapid for detection of neutralizing antibody than the conventional assay based on passaging infected cells, and more sensitive for detection of low-level CAV antibodies than a commercial blocking ELISA.

Animals↗
PubMed2000

Persistence of viral replication after anti-HBe seroconversion during antiviral therapy for chronic hepatitis B.

BACKGROUND/AIMS: Hepatitis B virus genome mutants may be selected during the immune-mediated clearance of infection or during long-term nucleoside analog administration and may escape both antiviral pressures. The pattern of anti-HBe seroconversion was analyzed in patients receiving new nucleoside analogs, lamivudine or famciclovir, in comparison with patients treated with interferon alpha. METHODS: Eighteen consecutive patients who seroconverted to anti-HBe were included in the study. Serial serum samples were studied with the quantitative determination of HBV DNA by the branched DNA assay (Chiron) and by a quantitative PCR assay (Roche diagnostics), determination of pre-S1 Ag, the genetic analysis of the viral genome with the determination of pre-core promoter or pre-core region mutations with a line probe assay (Innogenetics) and, in selected samples of polymerase gene mutations. RESULTS: The quantitative PCR assay was found to be more sensitive than the bDNA assay, allowing a 25-log decrease in viral DNA levels to be demonstrated after anti-HBe seroconversion. Viral persistence after anti-HBe seroconversion induced by interferon, lamivudine or famciclovir, was often associated with circulating HBV genomes harboring mutations in the precore promoter. The clinical significance of these findings was demonstrated by the observation of reversion to HBeAg in two patients treated with interferon and one with lamivudine. CONCLUSION: Persistence of significant levels of viremia that are not detected by the branched DNA assay may be observed after anti-HBe seroconversion. A precise monitoring of viremia levels with more sensitive assays and HBV mutant strains is warranted in patients undergoing antiviral therapy.

2-Aminopurine↗
PubMed1992

Selective expansion of T cells expressing T-cell receptor variable regions V beta 2 and V beta 8 in Kawasaki disease.

Kawasaki disease (KD) is an acute vasculitis complicated by the development of coronary artery abnormalities. The etiology of KD is unknown. Based on the observation that KD is associated with marked activation of T cells and monocyte/macrophages, we hypothesized that KD may be caused by a superantigen [e.g., a bacterial toxin that stimulates T cells expressing particular T-cell receptor beta chain variable (V beta) gene segments]. Peripheral blood T cells from patients in the acute and convalescent phases of KD and from various control groups were analyzed for T-cell receptor V beta gene expression by using a quantitative PCR technique and cytofluorographic analysis with available anti-V beta monoclonal antibodies. Patients with acute KD demonstrated significantly elevated levels of circulating V beta 2+ and V beta 8.1+ T cells compared to the other control groups. none of the other 20 V beta populations analyzed by quantitative PCR were found to be significantly elevated. Using flow cytometry, we confirmed a significant elevation of T cells reactive with anti-V beta 8.1 and the lack of change in several other V beta subsets--i.e. V beta 5.1, -5.2, -6.7, and -12. During the convalescence phase of KD, there was a reduction in the abnormal levels of V beta 2+ and V beta 8.1+ T cells. These observations suggest that KD may be caused by a superantigen and may provide insight into the nature of the etiologic agent.

Gene Expression↗
PubMed2006

Efficacy of HIV PCR techniques to diagnose HIV in infants born to HIV infected mothers--an Indian perspective.

AIM: To determine the efficiency of HIV DNA PCR (qualitative) and HIV RNA PCR (quantitative) to detect or rule out HIV infection in infants born to HIV infected mothers. SETTING: Pediatric and perinatal HIV clinic in a tertiary pediatric hospital. STUDY DESIGN: Prospective study METHODS AND MATERIALS: 52 infants born to HIV positive mother were tested for HIV infection by HIV DNA PCR or HIV-1 viral load from 1.5 to 7 months of age. Their HIV status was confirmed by an HIV ELISA test at 18 months of age. RESULTS: Of the 36 patients tested by HIV DNA PCR test, 15 patients (41.8%) had a positive test and 21 patients (58.2%) had a negative test. 12 patients (80%) had a false positive test (80%) and no patient had a false negative test. Sensitivity of HIV DNA test 100% with specificity of 53.9%. Of the 18 patients tested by HIV viral load, 17 patients (94.4%) had a negative test and 1 patient (5.6%) had a positive test. No patient had a false negative or false positive test. Sensitivity and specificity of HIV viral load was 100%, which was statistically significant (p=0.0004). CONCLUSION: HIV-RNA PCR (quantitative) is highly specific and sensitive test for diagnosing and excluding perinatal HIV infection in children in India.

DNA, Viral↗
PubMed2002

Quantification of feline herpesvirus 1 DNA in ocular fluid samples of clinically diseased cats by real-time TaqMan PCR.

A fluorogenic PCR was established for the quantification of feline herpesvirus 1 (FeHV-1) DNA in ocular fluid samples of clinically diseased cats. The new assay was specific for FeHV-1 and sensitive. The 100% detection rate ranged from 0.6 to 6 50% tissue culture infective doses per sample. When spiked samples with known quantities of virus were used, infectious virus titers and quantification of viral DNA by PCR correlated to each other in a linear fashion (R(2) = 0.9858) over a range of 4 orders of magnitude. Within this range, it was possible to calculate the FeHV-1 DNA content from a given infectious dose, and vice versa. The new diagnostic procedure was applied to ocular fluid samples from cats experimentally infected with FeHV-1 and specific FeHV-1-free cats. A good correlation between virus titer and quantitative PCR was observed, although only early in infection. In a second stage, the titer of infectious virus collapsed, while the PCR signal remained high. A constantly decreasing PCR signal accompanied by negative virus isolation was characteristic for a final stage of the infection. Finally, clinical samples from 20 cats that were suspected to suffer from FeHV-1 infection were analyzed. By comparing virus titers and quantitative PCR signals, it was possible to determine the current stage of the ongoing infection. Based on these findings, comparison of the results of consecutive samples allows the tracking of the course of the infection. Therefore, the new method combines the advantages of the two previously established conventional methods, qualitative PCR and virus isolation and titration.

Animals↗
PubMed2006

Measles viral load may reflect SSPE disease progression.

Subacute sclerosing panencephalitis (SSPE) is a rare, slowly progressive neurological disorder caused by the persistent infection with measles virus (MV). Despite much research into SSPE, its pathology remains obscure. We examined autopsy tissues of eight SSPE patients by real time quantitative PCR, immunohistochemistry and immunoblotting to determine viral load. MV N, M and H gene RNA could be detected in the central nervous system (CNS) of all patients and in two non-CNS tissues of one patient. The viral burden between patients differed up to four-fold by quantitative PCR and corresponded with detection of MV protein. The level of both viral RNA and antigen in the brain may correlate with disease progression.

Adolescent↗
PubMedDate not supplied

Reliability and discriminant validity of HER2 gene quantification and chromosome 17 aneusomy analysis by real-time PCR in primary breast cancer.

There is an increasing demand for the evaluation of HER2 status in breast cancer. In this study, sections from fixed tissues and triton extracts of tissue homogenates were obtained from 163 malignant breast tumors and analyzed in parallel using immunohistochemistry combined with fluorescence in situ hybridization, as gold standard tests, and an ELISA test (c-erbB2/c-neu Rapid Format ELISA, Oncogene Research Products, USA). Tumor DNA was employed to evaluate two quantitative PCR methods: the HER2/neu DNA Quantification Kit (Roche Diagnostics GmbH, Germany), which uses the gastrin chromosome 17 reference gene, and our recently developed Oncolab qPCR assay, where both a chromosome 17 gene (somatostatin receptor type II (SSTR2)) and a non-chromosome 17 reference gene (glyceraldehyde-3-phosphate deshydrogenase (GAPDH)) were used to detect an increase in HER2 gene copy number and to evaluate the aneusomy of chromosome 17, respectively. By IHC/FISH and ELISA, HER2 was overexpressed in 27 (16.6%) and 24 (14.7%) samples, respectively. With the Roche and Oncolab qPCR assays, 29 (17.8%) samples showed a ratio of HER2/gastrin > or = 2.0 and 26 (16.0%) showed a ratio of HER2/SSTR2 > or = 2.0, respectively. In samples presenting HER2/SSTR2 <2.0 and HER2/GAPDH > or = 2.0, which was indicative of a chromosome 17 polysomy, we observed a modest increase in HER2 protein expression. Complete agreement between the four methods for HER2 status determination was obtained for 154 (94.5%) samples. Overall, these results demonstrate that quantitative PCR is a reliable method for analyzing HER2 status and chromosome 17 polysomy.

Biomarkers, Tumor↗