Search PubMed⌕ Search

SEARCH · Search PubMed

Results for “Quantitative PCR”

Search indexed PubMed citations on genomics, clinical trials, systematic reviews and public health. Explore titles, authors and supplied subject terms, then open the PubMed record.

Quote a phrase for an exact phrase match. Source license links do not imply unrestricted reuse.

At least 505 records · Page 28Linked to original sources
PubMed1999

A quantitative PCR measurement of messenger RNA expression of IGF-I, IGF-II and IGFBP-5 in human skeletal muscle.

Insulin-like growth factor-I and -II (IGF-I and IGF-II) and their binding proteins are important components in growth promotion and tissue maintenance. We determined the presence of IGF-I, -II, and binding protein 5 (IGFBP-5) gene expression in human skeletal muscle and that mRNA abundance is not altered by nutrients and insulin. In the first protocol, (control) subjects were given water. In the second protocol, half of these subjects drank Polycose (carbohydrate) and the remaining subjects drank equal calories as a mixed meal. Quadriceps muscle biopsies were taken at 10 h. A semi-quantitative polymerase chain reaction was designed to measure gene expression. IGF-I, IGF-II and IGFBP-5 mRNA are present in adult human skeletal muscle, but no significant changes between meal groups were observed for IGF-I, IGF-II or IGFBP-5 mRNA levels, indicating that the expression of these genes are not altered acutely by nutrients and insulin.

Adult↗
PubMed2002

The role of quantitative PCR for the immune monitoring of cancer patients.

Human tumour immunology is at a standstill whereas implemented cancer vaccines have shown effectiveness in inducing immune responses detectable in circulating lymphocytes. In most circumstances, however, such immune responses are not sufficient to induce cancer regression. This paradoxical observation could be explained in several ways depending upon the immunological endpoint used for immune monitoring. For instance, analysis of immune responses in circulating lymphocytes that address the presence of T cells bearing T-cell receptors specific for the epitope used for vaccination, can accurately enumerate the number of T cells elicited by the vaccines but does not yield information about their functional status. Other monitoring strategies may yield general information about the reactivity of various T cells in response to a relevant stimulus and, therefore, may provide information more relevant to the purpose of the immunisation. Furthermore, the material used to monitor immune responses may, in itself, determine the significance of the findings obtained. In the assessment of the therapeutic efficacy of specific cancer treatment, analysis of immune responses in circulating lymphocytes (systemic response) may not be as relevant as the analysis of the same effector populations within the tumour microenvironment (peripheral response). This review will describe a novel approach that allows extreme flexibility in the analysis of systemic and peripheral responses by accurately measuring the level of expression of relevant genes using quantitative real-time reverse transcriptase polymerase chain reaction.

Animals↗
PubMed2000

Monitoring of human herpesvirus-6 and -7 genomes in saliva samples of healthy adults by competitive quantitative PCR.

Human herpesviruses-6 and -7 (HHV-6 and HHV-7) are thought to be transmitted during early infancy through saliva. However, the kinetics of the virus shedding in saliva of healthy adults, from whom children are assumed to acquire the viruses, is mostly unknown. This study was conducted to determine how many copies of the genome are secreted in saliva of healthy adults and to clarify the relationship between viral DNA load and virus isolation of HHV-6 and HHV-7. Competitive PCR was performed using primer sets in the U42 gene of each viral genome. In saliva samples from 29 healthy adults, HHV-6 and HHV-7 DNA was detected in 41.4% and 89.7%, respectively. The average copy number of the HHV-7 genome in the positive samples was higher than that of the HHV-6 genome. Follow-up studies of six seropositive individuals for 3 months showed that the amount of HHV-7 DNA was constant in each individual and that "high producers" and "low producers" could be distinguished. By contrast, the amount of HHV-6 DNA varied drastically over time in each individual. Although HHV-6 was never isolated from the saliva of any of the six individuals during the follow-up period, HHV-7 was isolated from each individual several times. The amount of HHV-7 DNA tended to be higher at the times when the virus was isolated than at the times when the virus was not isolated. These data demonstrate a striking contrast between HHV-6 and HHV-7 in the kinetics of genome and virus shedding.

Adult↗
PubMed1998

Over-expression of epidermal growth factor receptor and c-erbB2/neu but not of int-2 genes in benign prostatic hyperplasia by means of semi-quantitative PCR.

It is well established that activation of cellular genes may trigger uncontrolled cell growth and cancer development. Previous reports suggest that the epidermal growth factor receptor (EGFr), c-erbB2/neu and int-2, fibroblast growth factor-3, may be implicated in the development of benign prostatic hyperplasia (BPH). Using the polymerase chain reaction technique, we have assessed the amplification and expression of these molecular markers in 30 prostate samples from patients with BPH as well as from 5 normal donors. We detected mRNA over-expression of EGFr and c-erbB2/neu in 36% and 63%, respectively, of the BPH samples, but no gene amplification was found. No amplification or over-expression of int-2 was detected in any of the samples analyzed, suggesting that int-2 is not involved in BPH. Our results thus suggest a role for EGFr and c-erbB2/neu but not for int-2 in the development of BPH.

Aged↗
PubMedDate not supplied

Image analysis in quantitative PCR. An application for the measurement of c-erbB-2 oncogene amplification in DNA from human tumours.

We present an application of image analysis for the direct quantification of PCR products after gel electrophoresis and ethidium bromide staining of DNA. This procedure has been applied to the development of an assay based on competitive PCR for the measurement of the degree of amplification of c-erbB-2 oncogene in DNA from human tumours. In this method two DNA species (genomic and competitor) compete for PCR amplification. Since results are calculated from the final competitor/genomic ratio any variable affecting the rate of PCR amplification has no effect on the accuracy of the ratio measurement. Results are reported which show that even large variations in the experimental conditions (number of PCR cycles, sample volumes and extracted DNA quality) did not interfere with the precision of the measurement of the competitor/genomic ratio.

Binding, Competitive↗
PubMed2006

Rapid and accurate determination of MYCN copy number and 1p deletion in neuroblastoma by quantitative PCR.

MYCN amplification and 1p36 deletion are adverse prognostic factors in neuroblastoma, and rapid accurate determination of MYCN amplification is essential for risk stratification. MYCN copy number and 1p36 deletion status were determined by fluorescence in situ hybridization (FISH) and real time PCR in a diagnostic pathology laboratory setting on 35 consecutive patients with neuroblastoma. The PCR technique was technically successful in all cases and results were generally available within 24 hr of biopsy. There was no discordance between FISH and PCR results. Real time PCR is a reliable, accurate, and simple technique that can be applied to small neuroblastoma biopsies allowing rapid diagnosis.

Child, Preschool↗
PubMed2002

Real-time quantitative PCR for the detection of Streptococcus pneumoniae in the middle ear fluid of children with acute otitis media.

PCR based on the amplification of pneumolysin gene fragments has previously been applied to demonstrate Streptococcus pneumoniae in clinical specimens. Here, a real-time PCR method for the detection and quantification of pneumococci by amplifying a 206-bp fragment of the pneumolysin-encoding gene is described. The amplified fragments were detected simultaneously using fluorescent-labeled sequence-specific hybridization probes. The applicability of the assay to clinical samples was evaluated by studying 50 middle ear fluid (MEF) specimens from children with acute otitis media. Twenty-six of the MEF samples were positive by real-time PCR and the numbers of genome equivalents detected varied from 90 to 88,000/microl in 17 culture-positive samples and from 1 to 1,200/microl in 9 culture-negative samples. The results were compared to culture findings and to results obtained by using agarose gel electrophoresis or Europium-labeled hybridization probes for the detection of amplification products of conventional PCR. The sensitivity and specificity of the real-time PCR assay developed in the present study compared to culture were 100 and 73%, and to conventional PCR with agarose gel and/or TRF detection 93 and 96%, respectively. The real-time PCR assay was found to be rapid, easy to use, and sensitive in detecting and quantifying pneumococci.

Acute Disease↗
PubMed2001

An overview of real-time quantitative PCR: applications to quantify cytokine gene expression.

The analysis of cytokine profiles helps to clarify functional properties of immune cells, both for research and for clinical diagnosis. The real-time reverse transcription polymerase chain reaction (RT-PCR) is becoming widely used to quantify cytokines from cells, body fluids, tissues, or tissue biopsies. Being a very powerful and sensitive method it can be used to quantify mRNA expression levels of cytokines, which are often very low in the tissues under investigation. The method allows for the direct detection of PCR product during the exponential phase of the reaction, combining amplification and detection in one single step. In this review we discuss the principle of real-time RT-PCR, the different methodologies and chemistries available, the assets, and some of the pitfalls. With the TaqMan chemistry and the 7700 Sequence Detection System (Applied Biosystems), validation for a large panel of murine and human cytokines and other factors playing a role in the immune system is discussed in detail. In summary, the real-time RT-PCR technique is very accurate and sensitive, allows a high throughput, and can be performed on very small samples; therefore it is the method of choice for quantification of cytokine profiles in immune cells or inflamed tissues.

Animals↗
PubMed2025

Absolute Quantification of Cellular and Cell-Free Mitochondrial DNA Copy Number from Human Blood and Urinary Samples Using Real Time Quantitative PCR.

Mitochondrial DNA copy number (mtDNA-CN) in human body fluids is widely used as a biomarker of mitochondrial dysfunction in common metabolic diseases. Here we describe protocols to measure cellular and/or cell free (cf)-mtDNA-CN in human peripheral blood and urine. Cellular mtDNA is located inside the mitochondria where it encodes key subunits of the respiratory complexes in mitochondria and is usually normalized with reference to the nuclear genome as the mitochondrial genome to nuclear genome ratio (Mt/N) in either whole blood, peripheral blood mononuclear cells (PBMCs), or whole urine. Cf -mtDNA is usually found outside of the mitochondria, often released following mitochondrial damage, can trigger inflammatory pathways, and is usually measured as mtDNA-CN per volume of the starting material. Here we describe how to (1) separate whole blood into PBMCs, plasma, and serum fractions and whole urine into urinary supernatant and pellet, (2) prepare DNA from each of these fractions, (3) prepare reference standards for absolute quantification, (4) carry out qPCR for either relative or absolute quantification from test samples, (5) analyze qPCR data, and (6) calculate the sample size to adequately power studies. The protocol presented here is suitable for high throughput use and can be modified to quantify mtDNA from other body fluids, human cells, and tissues.

Humans↗
PubMed1994

A novel multispecific competitor fragment for quantitative PCR analysis of cytokine gene expression in rats.

Competitive polymerase chain reaction (PCR) is a sensitive method for quantification of cytokine mRNA expression. Co-amplification of an internal standard serves as control for comparing the efficiency of PCR in different samples. We have developed a novel control fragment for multiple analyses of rat cytokine gene expression containing primers for IL-1 beta, IL-2, IL-4, IL-5, IL-6, IL-10, TNF-alpha, TGF-beta 1, IFN-gamma and MIP-2. Additional primers were incorporated to analyse the content of T cells (CD3), activated T cells (CD25) and housekeeping genes (beta-actin and HPRT). As an example we demonstrate analysis of IL-2 mRNA expression in small pieces of kidney tissue obtained from rats after kidney allotransplantation. The IL-2 expression decreased tenfold during treatment with an anti-rat CD4 monoclonal antibody as compared to untreated animals.

Animals↗
PubMed1992

Characterization of HPV-16 E6/E7 transcription in CaSki cells by quantitative PCR.

Human papillomavirus (HPV) is associated with specific benign and malignant lesions of the epithelial and mucosal surfaces. Of the sexually transmitted types, HPV type 16 (HPV-16) is the most commonly associated with carcinoma of the uterine cervix. Expression of the E6/E7 open reading frame of the viral genome is considered critical in the development of neoplasia. Using the CaSki cervical carcinoma cell line as a model system, we have adapted the polymerase chain reaction to quantify the transcripts expressed from this region. It was found that 97.1% of the total spliced transcript is E6*I, which putatively encodes the E7 oncoprotein, while E6*II comprises 2.9% of spliced product. The ratio of E6*I to E6*II expression may be an important parameter in evaluating the disease risk associated with HPV-16 infection.

Base Sequence↗
PubMed2004

High-throughput detection of knockdown resistance in Myzus persicae using allelic discriminating quantitative PCR.

The peach-potato aphid Myzus persicae (Sulzer) has developed resistance to pyrethroid insecticides as a result of a mechanism conferring reduced nervous system sensitivity, termed knockdown resistance (kdr). This reduced sensitivity is caused by two mutations, L1014F (kdr) and M918T (super-kdr), in the para-type voltage-gated sodium channel. We have developed a diagnostic dose bioassay to detect kdr and provide preliminary information on the genotype present. We also developed two allelic discrimination PCR assays to determine precisely the genotypes of the two mutations (L1014F and M918T) in individual M. persicae using fluorescent Taqman MGB probes. In combination with assays for elevated carboxylesterase levels and modified acetylcholinesterase (MACE), this suite of assays allows for rapid high-throughput diagnosis, in individual aphids, of the three main resistance mechanisms of practical importance in the UK.

Alleles↗