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At least 487 records · Page 27Linked to original sources
PubMed1995

Mitochondrial DNA deletion analysis: a comparison of PCR quantitative methods.

The role of mitochondrial DNA (mtDNA) deletions in aging and in neurodegenerative diseases is often determined by measuring the amount of deleted mtDNA in the affected tissue. Upon examining brain autopsy tissue from a 59 year old individual with lung cancer we determined by serial dilution PCR and kinetic PCR that a greater ratio of deleted mtDNA was present in the caudate than in the parietal cortex. However, the magnitude difference for these two brain regions appeared to be technique dependent; by serial dilution PCR the caudate had 10 times more deleted mtDNA than the parietal cortex (0.0141 vs 0.0014) whereas kinetic PCR yielded a 4-fold difference (0.1258 vs 0.0316). These results indicate that although it is valid to compare the amount of deleted mtDNA in normal and diseased tissue and draw conclusions based on relative comparisons within one study, greater caution should be exercised when comparing absolute values from studies using different measurement techniques.

Base Sequence↗
PubMedDate not supplied

[A nonisotopic method of quantitative PCR analysis in diagnosing HIV infection].

A nonisotopic method for the detection of HIV DNA has been developed, based on the use of avidin-biotin complex to identify the products of polymerase chain reaction (PCR). Biotin label integrated in deoxyuridine triphosphate (bio-11-dUTP) was incorporated in the structure of amplified fragments in the course of PCR. The resultant products were identified in hybridization reaction with specific HIV DNA adsorbed on polystyrene plates. The suggested method was not inferior to the traditional radioisotope method in sensitivity and specificity and helped quantitatively assess the results of experiments using an objective criterion--optic density value.

Avidin↗
PubMed2005

Real-time quantitative PCR with gene probe, fluorochrome and flow cytometry for microorganism analysis.

Microorganism concentrations and viability can be better understood and clarified by using both culture and non-culture methods. Here, using pure suspensions of E. coli, three non-culture methods, namely, flow cytometry (FCM), epifluorescence microscopy (EFM), and real-time quantitative polymerase chain reaction (real-time qPCR), were compared with a traditional culture-based method. Using fluorocome-labeling methods with FCM and EFM applications, acridine orange (AO) and propidium iodide (PI) dyes were used to determine the total cell concentration and microorganism viability, respectively. The results indicated that total cell concentrations determined using FCM were statistically higher (2.62-4.94 times) than those determined using EFM. The difference might be due to cell losses induced by extensive preparations needed for EFM. In addition, EFM and FCM were highly associated for both the total cell concentration and viability. FCM-measured viability was the highest, whereas the culture-measured viability was the lowest. Furthermore, DNA concentrations measured by real-time qPCR with gene probe were highly associated with the total number concentrations measured by either the EFM or FCM. In summary, the three non-culture methods compared here could provide rapid and accurate information about microorganism concentrations and viabilities.

Acridine Orange↗
PubMed2001

Investigation of varicella-zoster virus DNA in lymphocyte subpopulations by quantitative PCR assay.

To investigate the nature of viremia during the acute phase of varicella, we studied the viral load in nine otherwise healthy children with varicella. Plasma and peripheral blood mononuclear cells (PBMC) were obtained, then PBMC were divided into CD4+T, CD8+T, and B lymphocytes and monocyte/macrophage fractions. The viral DNA in each component was quantified using a real-time quantitative polymerase chain reaction assay. Varicella-zoster virus (VZV) DNA was detected in plasma, PBMC and all subpopulations. The amount of viral DNA was similar in each PBMC subpopulation, suggesting that each lymphocyte fraction and monocytes carry similar amounts of VZV DNA during viremia.

Animals↗
PubMed2003

Rapid diagnosis of alpha(o)-thalassemia using the relative quantitative PCR and the dissociation curve analysis.

A method for diagnosis of alpha(o)-thalassemia was developed based on detection of accumulated PCR product using SYBR Green I, a double-stranded DNA binding dye, and a fluorescence-detecting thermocycler. Primers were designed to specifically amplify - -SEA and - -Thai deletions of alpha(o)-thalassemia. Albumin was selected as the reference gene. The comparative CT method was used to quantitate the target gene relative to the albumin gene. Dissociation curve analysis was used as a qualitative tool to distinguish different types of alpha(o)-thalassemia. In this study, the melting temperatures of the - -Thai and - -SEA deletions were 83 and 86 degrees C, respectively. Application of the assay for the diagnosis of alpha(o)-thalassemia heterozygotes and homozygotes is reported. The assay is highly robust and amenable to high throughput. It is thus a useful tool for the rapid detection of alpha(o)-thalassemia in populations with a high frequency of alpha(o)-thalassemia, - -SEA and - -Thai deletions.

Benzothiazoles↗
PubMed1993

Quantitative PCR for hepatitis B virus with colorimetric detection.

A novel, sensitive colorimetric test is described for quantification of the initial number of hepatitis B virus (HBV) genomes amplified in PCR. The viral genomes are amplified together with a synthetic internal standard (IS) to correct for the variability of the efficiency factor. One of the two primers is biotinylated, and the amplified mixtures of HBV and IS DNAs are bound to streptavidin-coated microtiter plates for quantitative detection. The ratio of HBV to IS DNA is determined for each sample by hybridization with DNP-containing probes and immunoenzymatic detection. The colorimetric detection is quantitative, rapid, and accurate with a dynamic range from approximately 10(8) to > 10(11) DNA molecules. The initial number of HBV genomes in a clinical sample is interpreted from the signal ratio HBV/IS by using a standard curve, obtained from coamplification of known quantities of synthetic HBV templates with IS. The assay quantified 15 viral genomes from 10 microliters of serum, and its dynamic range was up to five orders of magnitude. After the amplification step, the assay takes > 2 hr, and the method is applicable to automation.

Base Sequence↗
PubMed1997

Mixed hematopoietic chimerism after allogeneic bone marrow transplantation: the impact of quantitative PCR analysis for prediction of relapse and graft rejection in children.

It still remains unclear whether patients with mixed hematopoietic chimerism (MC) after allogeneic bone marrow transplantation (allo-BMT) have an increased risk of developing relapse or graft failure. To address this question, we monitored the individual dynamics of chimerism after allo-BMT in pediatric patients within a prospective case control study. The individual ratio of donor to recipient peripheral white cells was determined by quantification of genomic variable number of tandem repeats (VNTRs) with a polymerase chain reaction (PCR) approach. Within the study period from 1 January 1994 until 1 July 1996 we investigated 50 sequences of 46 pediatric patients after allo-BMT (32 with malignant, 18 with nonmalignant diseases). We found complete chimerism (CC) in 34/50 cases, MC in 12/50 follow-ups and 4/50 patients revealed autologous recovery (AC). Eight of 12 patients with MC showed increasing autologous patterns and subsequently relapsed or rejected their graft, 3/12 decreasing amounts of recipient DNA and turned to CC upon further follow-up. One patient of 12 who had severe combined immunodeficiency (SCID), attained engraftment with a stable MC pattern. Three patients of 34 with CC relapsed lacking a transitional MC interval. However, the time span between last CC confirmation and relapse in each of these three patients was 6 months or longer. We suggest that these patients also developed a stage of transitional MC but that the critical timepoint of molecular confirmation by PCR was missed as time intervals in the individual follow-up of these three patients were too long (> or = 6 months). In summary, the results demonstrate that the individual risk of developing relapse or graft failure is significantly enhanced in the MC situation (P < 0.0005). Therefore the quantitative analysis of MC at short time intervals might be of great value to identify high risk patients which will have a significantly/enhanced risk for relapse or graft rejection.

Adolescent↗
PubMed2001

Residual hepatitis B virus particles in liver transplant recipients receiving lamivudine: PCR quantitation of HBV DNA and ELISA of preS1 antigen.

Lamivudine, an antiviral agent, has a potential role in the treatment of recurrent or acquired de novo hepatitis B virus (HBV) infection after liver transplantation. During lamivudine therapy, residual HBV particles in serum, PBMC, and liver were quantified in 7 patients in whom hepatitis B occurred de novo (n = 4) or recurred (n = 3). HBV DNA and preS1 antigen were measured using a sensitive PCR technique and an in-house ELISA method, respectively. The genetic and antigenic properties of HBV variants that emerged during lamivudine treatment were also examined. One month after the outset of lamivudine treatment, all 7 patients remained positive for both HBV DNA and preS1 antigen in serum, reflecting residual HBV replication. At the end of therapy, four patients were considered to be lamivudine responders, including one who seroconverted to anti-HBs but remained HBV DNA positive in the liver (> 10(3) copies/microg of DNA). Among the three patients who did not respond to lamivudine, one had pol mutations (L450P and S550C) that had not been described previously, in addition to the common mutations within the YMDD locus and B domain. Defective core and preS viral proteins and atypical sedimentation profiles of HBV DNA-positive particles were observed in all three lamivudine-resistant patients. These findings confirm the persistence of HBV in liver transplant recipients despite strong inhibition of replication by lamivudine, and show abnormal viral transcription and/or morphogenesis in lamivudine-resistant patients.

Adult↗
PubMed2005

Real-time RT-PCR quantitative assays and postmortem degradation profiles of erythropoietin, vascular endothelial growth factor and hypoxia-inducible factor 1 alpha mRNA transcripts in forensic autopsy materials.

Recent advances in molecular biology have suggested the potential usefulness of mRNA analyses in postmortem investigations of fatal mechanisms. The aim of the present study was to establish quantitative assays of oxygen-regulated factors including erythropoietin (EPO), vascular endothelial growth factor (VEGF) and hypoxia-inducible factor 1 alpha (HIF1A) mRNAs, and to investigate the postmortem stability of these mRNA transcripts in forensic autopsy materials. Relative quantification of EPO, VEGF and HIF1A mRNAs, based on the TaqMan reverse transcription-polymerase chain reaction (RT-PCR), was performed on autopsy tissue specimens from the heart (n = 10), brain (n = 10), kidney (n = 16) and lung (n = 8) after preservation at room temperature for various storage times. VEGF and HIF1A mRNA gradually degraded in patterns similar to glyceraldehydes-3-phosphate dehydrogenase (GAPDH) mRNA used as an endogenous reference. Accordingly, the relative quantification of VEGF/GAPDH and HIF1A/GAPDH changed little up to 48h postmortem in tissue samples from the brain, kidney and lung except for a mild deviation of HIF1A in the myocardium. However, the status was different for EPO mRNA, with extraordinary stability for postmortem degradation and a marked postmortem time-dependent increase in the EPO/GAPDH ratio for all tissue samples. The present study suggested the potential for applying quantitative analyses of mRNA transcripts to autopsy materials and indicated the significance of investigating degradation profiles prior to carrying out relative quantification of target mRNAs in autopsy materials.

Brain Chemistry↗
PubMed1992

A simplified method for determination of specific DNA or RNA copy number using quantitative PCR and an automatic DNA sequencer.

Quantification of specific RNA or DNA molecules that are present in minute amounts in biological samples has previously been performed using PCR in the presence of an internal standard. We have adapted this concept by introducing several modifications that facilitate the quantification of the products and obviate the need for radioisotopes. After amplification, individual products are separated on sequencing gels and directly quantified using a fluorescent automated DNA sequencer. We describe two applications of this approach: the quantitation of minute amounts of bcr-abl hybrid mRNA from malignant cells and the determination of gene copy number in cells stably transfected with a plasmid bearing a chloramphenicol acetyltransferase gene.

Automation↗
PubMed1997

Quantitative PCR analysis of TNF-alpha and IL-1 beta mRNA levels in pediatric IBD mucosal biopsies.

Inflammatory bowel disease (IBD) is associated with increased activation of intestinal immune cells, whose overproduction of proinflammatory cytokines such as tumor necrosis factor-alpha (TNF-alpha) and interleukin-1 beta (IL-1 beta) is implicated in mediating the sustained inflammatory response. Studies to date have largely reported qualitative differences in cytokine gene expression between IBD and controls. Our aim was to perform quantitative analysis of intestinal mucosal mRNA expression in colonic biopsies from pediatric IBD patients using a competitive polymerase chain reaction. IL-1 beta and TNF-alpha were expressed in all IBD and control biopsies. Compared to controls, IL-1 beta mRNA levels were increased in involved tissue from Crohn's disease (CD) patients, but not in histologically uninvolved CD or in ulcerative colitis (UC) mucosa. IL-1 beta expression in the latter groups were equivalent to those found in tissue from patients with eosinophilic colitis (EOC). Significantly higher levels of IL-1 beta mRNA were found in uninvolved mucosa from CD patients who presented with a relapse of disease activity, as compared to newly diagnosed cases with histological features of CD at an early stage. TNF-alpha mRNA transcripts were also significantly elevated in involved CD mucosa, but not in the other groups. TNF-alpha gene expression in CD-involved tissue decreased with disease duration. Follow-up of the patients revealed that high cytokine expression in uninvolved CD tissue correlated with an early clinical relapse. In conclusion, quantitative determination of proinflammatory cytokine gene expression reveals differences between the type, severity, and clinical course in patients with IBD.

Adolescent↗
PubMed1993

Acidic and basic fibroblast growth factor mRNAs are increased in striatum following MPTP-induced dopamine neurofiber lesion: assay by quantitative PCR.

Acidic fibroblast growth factor (aFGF) and basic fibroblast growth factor (bFGF), the two best characterized members of a growing family of heparin-binding growth factors, have been shown to affect both survival of cultured neurons and regeneration of nerve terminals when applied exogenously. The endogenous expression of these growth factors in response to brain injury is not well understood. We have utilized the Swiss-Webster mouse, treated with the neurotoxin 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP), and a quantitative polymerase chain reaction assay to examine changes in endogenous synthesis of mRNA for both aFGF and bFGF in the striatum and substantia nigra. We have found that MPTP treatment results in a loss of 95% of dopaminergic function and is accompanied by an increase in expression of both aFGF and bFGF in the striatum at 1 week post-lesion. After 5 weeks, the terminals appear to be regenerating and FGF mRNA expression has returned to control levels. These results suggest that cellular reaction to chemical lesion in the brain may involve changes in growth factor expression, including both aFGF and bFGF.

1-Methyl-4-phenyl-1,2,3,6-tetrahydropyridine↗
PubMed2005

Quantitative PCR assay for cytochromes P450 2B and 3A induction in rat precision-cut liver slices: correlation study with induction in vivo.

INTRODUCTION: In drug development, new chemical entities that cause cytochrome P450 induction are considered to be undesirable since P450 induction is linked to tumor formation and may compromise the evaluation of drug safety when autoinduction results in poor drug exposure. METHODS: We evaluated the use of the precision-cut liver slice as a model for measuring induction of cytochrome P450 in rats. Quantitative real-time reverse-transcription polymerase chain reaction was used to analyze the induction of selected forms of cytochrome P450 at the mRNA level. Firstly, the system was validated against known inducers of CYP2B and 3A. Subsequently, 26 proprietary compounds were tested in rat liver slices and rats in vivo for CYP2B and 3A induction. RESULTS: Exposure of liver slices to the known CYP2B inducers phenobarbital, benzoyl-pyridine, cabarmazepine, metyrapone, RU486 and dexamethasone caused elevation of CYP2B1/2 expression 10- to 40-fold compared to the control values. The CYP3A inducers PCN, dexamethasone, nicardipine, nifedipine, clotrimazole and RU486 induced a 4- to 50-fold expression of CYP3A14. For 26 proprietary compounds, a correlation with an R(2) value of 0.74 was established between the induction of CYP2B expression in liver slices and that in rats in vivo. When liver slice results were used to predict the induction of CYP2B in rats in vivo, the success rate was 91%. The induction of CYP3A in rats in vivo was analyzed by Western blot, then quantified by densitometry. There was a good correlation between CYP3A induction in liver slices and induction in vivo as assessed by Western blot, with an 86% positive prediction rate. DISCUSSION: The use of liver slices in combination with TaqMan technology provides a good model for predicting CYP induction in the rat. This method is useful for identifying compounds with CYP2B and 3A induction liability in the early phase of drug discovery.

Algorithms↗
PubMed2004

Mitochondrial DNA 4,977-bp deletion in paired oral cancer and precancerous lesions revealed by laser microdissection and real-time quantitative PCR.

Oral cancer is the fourth leading cause of cancer deaths among men in Taiwan and is closely associated with areca quid chewing habits. Recent studies showed that mitochondrial DNA (mtDNA) mutations occur in various tumors, including oral cancers, and that the accumulation of mtDNA deletions could be an important contributor to carcinogenesis. Using laser microdissection, we have analyzed mtDNA deletions by pairwise comparisons in oral cancer, precancerous cells, and their adjacent submucosal stoma tissues in 12 patients with areca quid chewing history. Real-time quantitative polymerase chain reaction (RTQPCR) was performed to detect and quantify mtDNA with the 4,977-bp deletion in the histologically defined specified cell groups. Quantitative analysis of 60 samples by RTQPCR revealed that the average proportions of 4,977-bp deleted mtDNA over total mtDNA were 0.137%, 0.367%, and 0.001% in cancer, precancer cells, and lymphocytes of lymph node biopsies, respectively. Pairwise analysis of the proportion of mtDNA deletion in cancer, precancer, and their stroma tissues revealed a consistent trend among these patients. All of the patients (12/12) presented a higher proportion of mtDNA with 4,977-bp deletion in the lesions than in the lymphocytes, with average increases of 198-fold in cancer and 546-fold in precancer cells. A decrease in the proportion of deleted mtDNA was observed in 8 of 12 patients when the disease progressed from precancer to cancer lesions. Interestingly, 7 of 12 cancer tissues and 8 of 12 precancer lesions exhibited an average of 6.3-fold and 17.4-fold increases in the proportion of 4,977-bp deleted mtDNA in the stromal cells than in the lesion cells, respectively. The observation that the proportion of 4,977-bp deleted mtDNA in all oral lesions was higher than normal and consistently decreased during cancer progression from precancer to primary cancer suggests that accumulation and subsequent cytoplasmic segregation of the mutant mtDNA during cell division may play an important role in oral carcinogenesis. This study also demonstrates that laser microdissection combined with RTQPCR is an efficient and sensitive tool to gain insight into the role that mtDNA mutation may play in carcinogenesis.

Adult↗
PubMed1992

Measurement of mRNA by quantitative PCR with a nonradioactive label.

This report describes the development of a method to measure mRNA in small samples of human tissue by the polymerase chain reaction with a nonradioactive label. In this method RNA is reverse-transcribed in the presence of a control RNA, and subsequently amplified by the polymerase chain reaction during which a nonradioactive label (digoxigenin-11-dUTP) is incorporated. Gel blotting and immunological detection of digoxigenin followed by a chemiluminescent reaction provide an intense signal on film. This allows the detection and quantitation of 3-hydroxy-3-methylglutaryl (HMG) CoA reductase mRNA in 12 ng of RNA. We demonstrate that this is a sensitive and reproducible method, and that quantitation is linear with respect to the amount of mRNA present. The application of this method to the measurement of low density lipoprotein receptor and 3-hydroxy-3-methylglutaryl coenzyme A reductase mRNA levels in circulating peripheral blood mononuclear cells and human liver biopsy samples is discussed. The use of chemiluminescent reagents instead of radioactive labels allows this procedure to be performed safely in laboratories not equipped for radioactivity.

Base Sequence↗
PubMed2000

Detection of methylthioadenosine phosphorylase (MTAP) and p16 gene deletion in T cell acute lymphoblastic leukemia by real-time quantitative PCR assay.

Methylthioadenosine phosphorylase (MTAP) deficiency in tumors can be therapeutically exploited for selective therapy. Many tumors lacking MTAP have been found to homozygously delete the chromosome 9p region containing the p16 tumor suppressor gene. Several methods have been used to detect chromosome 9p deletions in primary tumors. However, the accurate diagnosis of chromosome 9p deletions has been hampered by the presence of contaminating normal cells. In search of an accurate and sensitive diagnostic method, we have developed the real-time polymerase chain reaction assay using the TaqMan chemistry for quantitative detection of MTAP and p16 gene deletions. The assay's feasibility was tested with peripheral blood leukocytes (PBL) from 29 patients with adult T cell leukemia (ATL) previously analyzed with Southern blot analysis and validated on 39 PBL or bone marrow samples from childhood T cell acute lymphoblastic leukemia (T-ALL). Homozygous deletions of MTAP and p16 genes were detected respectively in six (20.7%) and eight (27.6%) of 29 ATL samples and in 15 (38.5%) and 23 (59%) of 39 T-ALL samples. The results correlated well with those of Southern blot analysis. It is of significance that the newly developed method can successfully detect homozygous deletions of these genes in samples containing as low as 33% blast cells. This rapid and sensitive method may be useful in searching for candidates for selective therapy targeting MTAP deficiency.

Adult↗
PubMed1999

Development and evaluation of quantitative-competitive PCR for quantitation of coxsackievirus B3 RNA in experimentally infected murine tissues.

A method is described for quantitation of enterovirus RNA in experimentally infected murine tissues. Viral RNA was extracted from tissue samples and amplified by reverse transcriptase PCR in the presence of an internal standard RNA. The ratio of PCR product derived from viral RNA and internal standard RNA was then determined using specific probes in a post-PCR electrochemiluminescent hybridization assay. This provided an estimate of the viral RNA copy number in the original sample, and detection of PCR product derived from internal standard RNA validated sample processing and amplification procedures. RNA copy number correlated with viral infectivity of cell culture-derived virus, and one tissue culture infective dose was found to contain approximately 10(3) genome equivalents. The ratio of RNA copy number to infectivity in myocardial tissue taken from mice during the acute phase of coxsackievirus B3 myocarditis was more variable ranging from 10(4)-10(7), and was dependent on the stage of infection, reflecting differential rates of clearance for viral RNA and viral infectivity. The assay is rapid, and could facilitate investigations which currently rely upon enterovirus quantitation by titration in cell culture. This would be useful for experimental studies of viral pathogenesis, prophylaxis and antiviral therapy.

Acute Disease↗
PubMed2005

Real-time quantitative PCR analysis of gene dosages reveals gene amplification in low-grade oligodendrogliomas.

Proto-oncogene amplification is an important alteration that is present in about 45% to 50% of high-grade human gliomas. We studied this mechanism in 8 genes (cyclin-dependent kinase-4 [CDK4], MDM2, MDM4, renin-angiotensin system-1, ELF3, GAC1, human epidermal growth factor receptor-2, and platelet-derived growth factor receptor-A gene) in a series of 40 oligodendrogliomas (World Health Organization (WHO) grade II, 21; WHO grade III, 13; and WHO grade II-III oligoastrocytomas, 6) using real-time quantitative polymerase chain reaction. Amplification of at least 1 of these genes was detected in 58% of samples (23/40). By histopathologic grade, 67% of grade II oligodendrogliomas (14/21), 46% of grade III anaplastic oligodendrogliomas (6/13), and 50% of mixed oligoastrocytomas (3/6) were positive for amplification of at least 1 gene. CDK4, MDM2, and GAC1 were the most frequently involved genes (12/40 [30%], 12/40 [30%], and 13/40 [33%], respectively). Our findings demonstrate gene amplification in low-grade samples indicating that it is an important alteration in the early steps of oligodendroglioma development and, therefore, might be considered a molecular mechanism leading to malignant progression toward anaplastic forms.

Biomarkers, Tumor↗