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Results for “quantitative polymerase chain reaction”

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At least 19 recordsLinked to original sources
PubMed1993

5-Fluorouracil alters dihydrofolate reductase pre-mRNA splicing as determined by quantitative polymerase chain reaction.

A quantitative polymerase chain reaction (PCR) assay has been developed to determine the absolute and relative amounts of each dihydrofolate reductase RNA species present at different stages of splicing (i.e., pre-mRNA, splicing intermediate, and mRNA). The ratios of each RNA species as measured by quantitative PCR have been confirmed by S1 nuclease mapping analysis. Quantitative PCR studies reveal a concentration-dependent decrease in the levels of dihydrofolate reductase mRNA and a splicing intermediate but little change in pre-mRNA levels after long term exposure of cells to 5-fluorouracil (FUra). The observed changes correlate with the extent of FUra incorporation into RNA and with cytotoxicity. These results, together with previous data from our laboratory, provide the first direct evidence that FUra incorporation into RNA can cause inhibition of pre-mRNA splicing in vivo. Inhibition of pre-mRNA splicing is thus a likely additional mechanism by which FUra incorporation into RNA may lead to growth inhibition and cell death.

Base Sequence↗
PubMed1996

Theoretical uncertainty of measurements using quantitative polymerase chain reaction.

Current quantitative polymerase chain reaction (PCR) protocols are only indicative of the quantity of a target sequence relative to a standard, because no means of estimating the amplification rate is yet available. The variability of PCR performed on isolated cells has already been reported by several authors, but it could not be extensively studied, because of lack of a system for doing kinetic data acquisition and of statistical methods suitable for analyzing this type of data. We used the branching process theory to simulate and analyze quantitative kinetic PCR data. We computed the probability distribution of the offspring of a single molecule. We demonstrated that the rate of amplication has a severe influence on the shape of this distribution. For high values of the amplification rate, the distribution has several maxima of probability. A single amplification trajectory is used to estimate the initial copy number of the target sequence as well as its confidence interval, provided that the amplification is done over more than 20 cycles. The consequence of possible molecular fluctuations in the early stage of amplification is that small copy numbers result in relatively larger intervals than large initial copy numbers. The confidence interval amplitude is the theoretical uncertainty of measurements using quantitative PCR. We expect these results to be applicable to the data produced by the next generation of thermocyclers for quantitative applications.

Biometry↗
PubMed1994

DNA damage by anticancer agents and its repair: mapping in cells at the subgene level with quantitative polymerase chain reaction.

The quantitative polymerase chain reaction (QPCR)-based assay was used to measure DNA damage and repair to a small (523 bp) fragment of the single-copy human N-ras gene in K562 cells. Compared with previous methods DNA preparation from treated cells and the subsequent detection of the radioactive product were considerably simplified. The results demonstrated that QPCR can be used to measure damage in a small gene segment, caused by cisplatin, nitrogen, and quinacrine mustards. Drug-DNA adducts produced by two novel minor groove binding, sequence-specific molecules (AT-486 and DSB-120) could be detected at physiologically relevant concentrations of drug. For both cisplatin and nitrogen mustard the concentrations required to cause damage in cells were higher than those needed to cause equivalent damage in isolated DNA. In contrast both AT-486 and quinacrine mustard caused more damage at equimolar concentrations in cells than in isolated DNA. DSB-120, which is closely related to AT-486, was found to be 15-fold less effective than the latter at causing damage in treated cells despite similar reactivity with isolated DNA. Repair of damage caused by quinacrine mustard to the same small gene fragment was found to proceed at a constant rate over 24 h. The QPCR assay presented here is a simple quantitative method to measure damage and repair in subgene functional units such as promoters, introns, and exons.

Antineoplastic Agents↗
PubMed1996

Basics of quantitative polymerase chain reaction: 2. Electrophoresis and quantitation of polymerase chain reaction products.

The performance of agarose and polyacrylamide (PAGE) gels in quantitating polymerase chain reaction (PCR) amplified c-erbB2 and p53 gene sequences (213 and 133 base pairs, respectively) was studied by applying image analysis on photographed ethidium bromide stained gels. The 5 mm thick agarose gels were more insensitive than the 1 mm thin PAGE gels and already started to show saturation effects within a narrow concentration range. The explanation is the greater dilution of band-associated products within the volume of the gel and the inefficient penetration of exciting UV light through the gel. Such effects produced inconsistency and dramatic variations in the band ratio estimates. In the system used by us, agarose electrophoresis and also electrophoresis using 5 mm thick PAGE gels have only a limited value in quantitation of PCR products with the band density ratios, but both can be used well in qualitative work. The far thinner (1 mm) PAGE gels, which did not markedly absorb UV light, performed better in the light of band density ratio estimates. The linear or approximately linear range of concentrations was wider. The coefficient of variation of band ratio, when estimated from the same gels, after loading them with aliquots of identical DNA from several PCR amplified tubes, was in the range of 4%. The absolute values of band densities had a more remarkable variation (than 4%) in both agarose and PAGE gels. Our recommendation is that quantitation of PCR products be done with band density ratio estimates, and in thin PAGE gels.

Base Sequence↗
PubMed1992

Use of quantitative polymerase chain reaction to quantitate cytokine messenger RNA molecules.

A quantitative polymerase chain reaction (PCR) using an internal control (Standard) RNA was developed for precise quantitation of human cytokine mRNA. The target mRNA and internal control were simultaneously reverse transcribed and co-amplified using the same set of primers. The amount of specific target mRNA is then quantitated by extrapolating against the standard curve generated with the internal standard. The internal control RNA consisted of linearly connected sequences of the 5' primers of multiple cytokine genes followed by the complementary sequences to their 3' primers in the same order. This structure of the internal standard enables one to use the same internal standard for quantitating multiple cytokine mRNAs. Using this approach, we estimated the induced levels of cytokine mRNA, IL2, IL6, and TNF-alpha to be 2.8 x 10(6), 2.4 x 10(5) and 3.4 x 10(8) molecules per 1.0 microgram total cellular RNA of Jurkat, THP-1 and HL 60 cells, respectively. Comparable values were obtained when quantitation experiments were done on another batch of THP-1 and HL-60 total cellular RNA. The excellent sensitivity and reproducibility makes this approach a valuable one in following changes in cytokine gene expression in wide variety of conditions, both in vivo and in vitro.

Actins↗
PubMed2003

Persistent over-expression of specific CC class chemokines correlates with macrophage and T-cell recruitment in mdx skeletal muscle.

Prior studies and the efficacy of immunotherapies provide evidence that inflammation is mechanistic in pathogenesis of Duchenne muscular dystrophy. To identify putative pro-inflammatory mechanisms, we evaluated chemokine gene/protein expression patterns in skeletal muscle of mdx mice. By DNA microarray, reverse transcription-polymerase chain reaction, quantitative polymerase chain reaction, and immunoblotting, convergent evidence established the induction of six distinct CC class chemokine ligands in adult MDX: CCL2/MCP-1, CCL5/RANTES, CCL6/mu C10, CCL7/MCP-3, CCL8/MCP-2, and CCL9/MIP-1gamma. CCL receptors, CCR2, CCR1, and CCR5, also showed increased expression in mdx muscle. CCL2 and CCL6 were localized to both monocular cells and muscle fibers, suggesting that dystrophic muscle may contribute toward chemotaxis. Temporal patterns of CCL2 and CCL6 showed early induction and maintained expression in mdx limb muscle. These data raise the possibility that chemokine signaling pathways coordinate a spatially and temporally discrete immune response that may contribute toward muscular dystrophy. The chemokine pro-inflammatory pathways described here in mdx may represent new targets for treatment of Duchenne muscular dystrophy.

Animals↗
PubMed1997

Identification of Deletion Carriers in Duchenne/Becker Muscular Dystrophy Families Using a Digoxigenin-Labeled Quantitative Polymerase Chain Reaction Technique.

Background: A quantitative polymerase chain reaction (PCR) technique based on the incorporation of digoxigenin (DIG), and visualization of the labeled fragments for the detection of deletion carriers in Duchenne/Becker muscular dystrophy families has been developed. Methods and Results: Sixty-five DNA samples taken from mothers and/or sisters of familial and sporadic deletion patients were investigated in the exponential phase of amplification. All obligate carriers were correctly identified using this technique. In more than 95% of deletion families, possible carriers could be screened by using four different multiplex systems specifically designed to increase the efficiency of the detection. Deletions were found to be present in 42% of possible carriers. All these results were confirmed by computer-assisted laser densitometry. Conclusions: Dosage analysis by DIG-labeled quantitative PCR is a reliable and accurate technique for detecting Duchenne/Becker muscular dystrophy deletion carriers.

Journal Article↗
PubMed2003

Real-time polymerase chain reaction quantitation of relative expression of genes modulated by p53 using SYBR Green I.

Real-time quantitative polymerase chain reaction (QPCR) using the Roche LightCycler was used to verify the expression of asparagine synthetase (ASNS) identified by microarray analysis as a target of p53 transrepression and mutant p53 transactivation. A p53-null cell line derived from lung carcinoma, H1299, was infected with recombinant adenovirus expressing wild-type (WT) p53, mutant p53-D281G, or beta-galactosidase as a control. After 24 h of infection, RNA was harvested and used for microarray analysis. ASNS was one of several genes whose expression was down-regulated by WT p53 and up-regulated in the presence of mutant p53. Expression levels of ASNS were measured relative to an exogenously applied quality-control nucleic acid template. Real-time PCR product accumulation was monitored using the intercalating dye, SYBR Green I, which exhibits a higher fluorescence upon binding of double-stranded DNA. Relative gene expression was calculated using conditions at the early stages of PCR, when amplification was logarithmic and, thus, could be correlated to initial copy number of gene transcripts. ASNS was found to be down-regulated in the presence of WT p53 and up-regulated by mutant p53.

Benzothiazoles↗
PubMed2006

A robust approach for the quantitation of viral concentration in an adenoviral vector-based human immunodeficiency virus vaccine by real-time quantitative polymerase chain reaction.

A real-time quantitative polymerase chain reaction (PCR)-based method was developed to measure the concentration of recombinant adenoviral vector genomes in purified virus bulks and final container samples of monovalent and multivalent human immunodeficiency virus (HIV) adenoviral vector vaccine candidates. This method, referred to as the genome quantitation assay (GQA), was optimized through a rigorous approach for evaluating PCR detection chemistries, designing a robust assay format, and establishing a properly calibrated reference standard. In addition, the use of a simplified lysis procedure, automated liquid transfer system, and parallel-line data analysis contribute to an accurate, precise, reliable, and high-throughput assay procedure that can be used for process monitoring, final formulation, and release of vaccine products. A variance component analysis study indicated that the GQA typically produces results with an interassay precision of less than 10% relative standard deviation (RSD), allowing generation of final results (average of three runs) with associated interassay precision of 6% RSD or less. The precision, accuracy, specificity, and robustness of the GQA demonstrate its utility for analytical characterization of a wide variety of viral vector- and DNA plasmid- based vaccines or gene therapy products. In addition, we also evaluated the Adenovirus Reference Standard generated by the Adenovirus Reference Material Working Group in the GQA to provide a common point-of-reference for our analytical method.

AIDS Vaccines↗
PubMed1996

Analysis of cAMP RI alpha mRNA expression in breast cancer: evaluation of quantitative polymerase chain reaction for routine use.

A quantitative polymerase chain reaction (PCR) method for determining concentrations of mRNA for the cyclic AMP (cAMP)-binding protein RI alpha, a regulatory subunit of cAMP-dependent protein kinase, was developed using site-directed mutagenic primers and mix-melt PCR. The PCR product for RI alpha mRNA was modified to include an EcoRV restriction site for use as an internal standard. This mutant utilised the same primers as the target mRNA and differed in sequence by only four bases. As only one of these base changes results in a purine/pyrimidine switch the effective change in labelling with [32P]dCTP was less than 0.5%. Reverse transcription of mRNA was performed and quantitative PCR was carried out using fixed levels of mutant RI alpha vs varying amounts of both normal RI alpha sequence of known concentration and unknown samples. Validation of the technique using rigourous quality control established that reverse transcription, determined by incorporation of labelled nucleotides, gave intra- and interassay variations of 16.2 and 9.3% respectively. Using crossover evaluation of cDNA concentrations with cloned RI alpha sequences as controls intra- and interassay variations of 14.3% and 4-8% respectively were obtained. Using compounded errors, the limits of precision for this technique demonstrate that values that are altered by 50% or more represent a true alteration in mRNA levels between samples tested. This value compares favourably to similar values for radioimmunoassays of between 10% and 30% precision. Analysis of a series of patient samples during routine follow-up of treatment demonstrated clear changes in mRNA levels. Using site-directed mutagenesis to establish a quantitative PCR-based assay for expression of mRNA this study demonstrates the potential usefulness and some limitations of quantitative PCR for applications within a clinical biochemistry laboratory. However, based on compounded error, values that vary by less than 50% within assays, and by less than 70% in separate assays could not be clearly separated. Assessment of paired patient samples has demonstrated clear changes in mRNA for the target protein RI alpha. With the use of normal quality control procedures this study has established that the degree of reproducibility of this quantitative PCR technique would allow assessment of mRNA levels for markers of interest in clinical samples in a routine laboratory context.

Base Sequence↗
PubMed2004

Immuno-quantitative polymerase chain reaction for detection and quantitation of prion protein.

Immuno-polymerase chain reaction (PCR) is an extremely sensitive detection method, combining the specificity of antibody detection and the sensitivity of PCR. We have developed an immuno-quantitative PCR (iqPCR), exploiting real-time PCR technology, in order to improve this immuno-detection method and make it quantitative. To illustrate the advantages of iqPCR, we have compared it with a conventional enzyme linked immuno sorbent assay (ELISA) technique in experiments aimed at detecting the cellular and the resistant form of prion protein in bovine brain extract. The iqPCR technique proved to be more sensitive than ELISA, so it could be a technique of choice for the diagnosis of infected animals both at an ante mortem and post-mortem stage.

Animals↗
PubMed2005

Evaluation of a real-time quantitative polymerase chain reaction assay for detection and quantitation of virulent Rhodococcus equi.

OBJECTIVE: To evaluate a real-time quantitative polymerase chain reaction (QPCR) assay in the detection and quantitation of virulent Rhodococcus equi. SAMPLE POPULATION: 1 virulent, 2 intermediately virulent, and 2 avirulent strains of R. equi and 16 isolates of bacteria genetically related to R. equi. PROCEDURE: The QPCR assay was evaluated for detection and quantitation of the virulence-associated gene (vapA) of R. equi in pure culture and in samples of tracheobronchial fluid, which were inoculated with known numbers of virulent R. equi. Results were compared with those derived via quantitative microbial culture and standard polymerase chain reaction methods. RESULTS: The QPCR assay detected the vapA gene in pure culture of R. equi and in tracheobronchial fluid samples that contained as few as 20 CFUs of virulent R. equi/mL and accurately quantitated virulent R. equi to 10(3) CFUs/mL of fluid. The assay was highly specific for detection of the vapA gene of virulent R. equi and was more sensitive than standard polymerase chain reaction for detection of R. equi in tracheobronchial fluid. CONCLUSIONS AND CLINICAL RELEVANCE: The QPCR assay appears to be a rapid and reliable method for detecting and quantitating virulent R. equi. The accuracy of the QPCR assay is comparable to that of quantitative microbial culture. The increased sensitivity of the QPCR method in detection of virulent R. equi should facilitate rapid and accurate diagnosis of R. equi pneumonia in foals.

Actinomycetales Infections↗
PubMed2003

Quantitative assessment of contaminating tumor cells in autologous peripheral blood stem cells of B-cell non-Hodgkin lymphomas using immunoglobulin heavy chain gene allele-specific oligonucleotide real-time quantitative-polymerase chain reaction.

A real-time quantitative-polymerase chain reaction (RQ-PCR) targeting the immunoglobulin heavy chain (IgH) gene has been used for the quantification of minimal residual disease (MRD) in B-cell hematological malignancies. In non-Hodgkin lymphoma (NHL), experimental costs are increased, as a large number of primer-probe sets are required because of diversity, due to somatic and ongoing mutations of the IgH gene. We developed an allele-specific oligonucleotide (ASO) combined with a germline consensus probe-based RQ-PCR assay and examined MRD in peripheral blood stem cells (PBSC). The IgH consensus probes were adapted in seven (50%) of 14 amplifiable cases. Patients with heavily contaminating tumor cells in PBSC relapsed after PBSC transplantation. Our strategy will contribute to the development of a cost-efficient, precisely quantitative and systemic detection assay for MRD in NHL.

Adolescent↗
PubMed2005

[Software and hardware design for the temperature control system of quantitative polymerase chain reaction].

A temperature control system for quantitive polymerase chain reaction (PCR) is presented in the paper with both software and hardware configuration. The performance of the control system has been improved by optimizing the software and hardware design according to the system's properties. The control system has been proven to have a good repeatability and reliability as well as high control precision.

Equipment Design↗
PubMed1993

cDNA equalization for reverse transcription-polymerase chain reaction quantitation.

Reverse transcription coupled with the polymerase chain reaction has been increasingly utilized to study gene expression. However, most previously published quantitative techniques are limited by accurate initial RNA quantitation and do not account well for the relative efficiency of reverse transcription. We have developed a technique of labeling and quantitating the random-primed cDNA product of a reverse transcription reaction. Using the polymerase chain reaction in conjunction with template dilutions or with an internal competitive template, we show that by normalizing the cDNA input into the polymerase chain reaction, we get accurate quantitation of gene expression. We call this procedure cDNA equalization of reverse transcriptase-polymerase chain reaction. This method is ideal for small clinical samples where accurate quantitation of input RNA is difficult.

Animals↗
PubMed1996

Measurement of human herpesvirus 7 load in peripheral blood and saliva of healthy subjects by quantitative polymerase chain reaction.

Qualitative and competitive-quantitative nested polymerase chain reaction (PCR) assays were developed for human herpesvirus 7 (HHV-7). These assays amplify a DNA sequence encoding part of the HHV-7 homologue of the human herpesvirus 6 (HHV-6) U42 gene. The PCR assays were used to analyze peripheral blood DNA (pbDNA) and saliva from 24 healthy volunteers. The prevalence of HHV-7 in saliva was 96%, with a median virus load of 1.1 x 10(6) copies/mL. Longitudinal analysis revealed sustained virus load, suggesting continued active viral replication. Analysis of 1 microgram of pbDNA showed the prevalence of HHV-7 to be 83%, with a median virus load of 40 copies (267 copies/10(6) cells). Analysis of sequential pbDNA samples showed individuals to have stable levels of HHV-7 virus load. These data demonstrate persistence of HHV-7 at two distinct sites and provide baseline data allowing comparisons with HHV-7 load in immunocompromised patients.

Base Sequence↗
PubMed2003

Comparison of two quantitative polymerase chain reaction methods for detecting HER2/neu amplification.

Two quantitative polymerase chain reaction (PCR) methods for HER2/neu gene quantification were evaluated for implementation into a clinical laboratory. Assays were developed using sequence-specific hybridization probes to detect a target (HER2/neu) and a reference gene (beta-globin) simultaneously. One method utilizes real-time quantification while the second uses internal competitors and melting curves to quantify the unknown sample. These two methods were evaluated using three cell lines and 97 breast tumor samples. Two hundred ninety-four samples were subsequently evaluated using the real-time quantification and immunohistochemical (IHC) staining. Real-time PCR gave HER2/neu gene doses of 10 for SKBR3 and 2 for T47D while the competitive PCR gave doses of 11 for SKBR3 and 2.2 for T47D. Both methods produced coefficients of variation (CV) of less than 3% for within-run and less than 6% for between-run analysis. Examination of 97 breast tumors found a correlation of r = 0.974 between the two methods. IHC and PCR results agreed for 234 of the subsequent 294 samples analyzed (79% concordance). A subset of ten discrepant samples was microdissected. After microdissection all ten were positive by PCR, thus resolving the discrepancy. Real-time quantification and microdissection is useful clinically for HER2/neu quantification. Its ease of use and broad dynamic range allows screening for amplification of HER2/neu.

Base Sequence↗
PubMed2002

Quantitative assessment of hematopoietic chimerism after bone marrow transplantation by real-time quantitative polymerase chain reaction.

We have developed a real-time quantitative polymerase chain reaction (PCR) assay using TaqMan technology (Applied Biosystems, Foster City, CA) for monitoring donor cell engraftment in allogenic hematopoietic stem cell transplant recipients. For this purpose, we selected 19 specific sequence polymorphisms belonging to 11 human biallelic loci located on 9 different chromosomes. Using a set of specially designed primers and fluorogenic probes, we evaluated the 19 markers' informativity on a panel of 126 DNA samples from 63 recipient/donor pairs. In more than 90% of these pairs, discrimination between recipient and donor genetic profile was possible. By using serial dilutions of mixed DNAs, we evaluated the linearity and sensitivity of the method. A linear correlation with r higher than 0.98 and a sensitivity of 0.1% proved reproducible. Fluorescent-based PCR of short tandem repeats (STR-PCR) and real-time PCR chimerism assay were compared with a panel of artificial cell mixtures. The main advantage of the real-time PCR method over STR-PCR chimerism assays is the absence of PCR competition and plateau biases, and results evidenced greater sensitivity and linearity with the real-time PCR method. Furthermore, different samples can be tested in the same PCR run with a final result in fewer than 48 hours. Finally, we prospectively analyzed patients who received allografts and present 4 different clinical situations that illustrate the informativity level of our method. In conclusion, this new assay provides an accurate quantitative assessment of mixed chimerism that can be useful in guiding early implementation of additional treatments in hematopoietic stem cell transplantation.

Adolescent↗