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PubMed2003

Expression of galectin-3 in normal and malignant thyroid tissue by quantitative PCR and immunohistochemistry.

Galectin-3 has been extensively studied as an immunohistochemical marker of thyroid malignancy, and a high diagnostic accuracy has been reported even for difficult pathological diagnoses, such as minimal invasive follicular carcinoma. We consequently hypothesized that the quantitative analysis of galectin-3 mRNA rather than the more observer-dependent immunohistological determination might enhance the diagnostic workup of ambiguous thyroid lesions. In the present study, we set out to validate this approach by analyzing concomitantly the expression and production of galectin-3 in benign and malignant thyroid tumors by means of quantitative PCR and immunohistochemistry. Twenty-eight benign and 31 malignant thyroid samples were quantified by real-time PCR for the mRNA levels of galectin-3 and thyroglobulin. Galectin-3 protein expression was examined by immunohistochemistry in 13 benign and 14 malignant thyroid samples. There was a significant increase in galectin-3 at both the mRNA (12/20) and protein levels in papillary cancer (8/8), although the mRNA values overlapped partly with benign lesions. Surprisingly, only a focal and discrete galectin-3 immunoreactivity was seen in follicular cancer (1/5); no augmentation of the mRNA was found. The expression of the thyroid-specific gene thyroglobulin was highly variable in benign and malignant thyroid tissue. These results suggest that the quantitative measurement of galactin-3 mRNA is unlikely to be clinically useful and underscore the need for searching for novel markers for thyroid malignancies.

Adenocarcinoma, Follicular↗
PubMed2005

[Detection of duplications or deletions of the PMP22 gene using real-time quantitative PCR].

OBJECTIVE: To detect the duplication or deletion of peripheral myelin protein 22(PMP22) gene in Chinese patients with Charcot-Marie-Tooth disease(CMT) or hereditary neuropathy with liability to pressure palsies(HNPP) using real-time quantitative polymerase chain reaction. METHODS: Duplications or deletions of PMP22 gene were detected in 113 CMT cases, 4 HNPP cases and 50 normal controls by using real-time quantitative PCR. RESULTS: Thirty-six of 113 CMT cases had the PMP22 duplication, 4 HNPP cases had the PMP22 deletion. No duplication or deletion was found in 50 normal controls. CONCLUSION: The PMP22 duplication rate in Chinese patients with CMT is 31.9%(36/113). PMP22 deletion is the common cause of HNPP.

Adult↗
PubMed2003

Real-time quantitative PCR of Epstein-Barr virus BZLF1 DNA using the LightCycler.

Epstein-Barr virus (EBV) is associated with the development of lymphoproliferative disorders in the immunocompromised host and in particular post-transplant recipients. The development of a rapid real-time quantitative PCR for the detection of the immediate early gene BZLF1 is described using the Roche LightCycler. BZLF1 codes for the ZEBRA protein, one of the cascade of transactivation factors involved in the lytic life cycle of the virus. The assay is rapid and can detect a single copy of EBV DNA. Intra- and inter-assay variability studies using the external quantitative standards and DNA extracted from whole blood and plasma samples were shown to be within a 0.5 log(10) range.

DNA, Viral↗
PubMed2002

A quantitative PCR (TaqMan) assay for pathogenic Leptospira spp.

BACKGROUND: Leptospirosis is an emerging infectious disease. The differential diagnosis of leptospirosis is difficult due to the varied and often "flu like" symptoms which may result in a missed or delayed diagnosis. There are over 230 known serovars in the genus Leptospira. Confirmatory serological diagnosis of leptospirosis is usually made using the microscopic agglutination test (MAT) which relies on the use of live cultures as the source of antigen, often performed using a panel of antigens representative of local serovars. Other techniques, such as the enzyme linked immunosorbent assay (ELISA) and slide agglutination test (SAT), can detect different classes of antibody but may be subject to false positive reactions and require confirmation of these results by the MAT. METHODS: The polymerase chain reaction (PCR) has been used to detect a large number of microorganisms, including those of clinical significance. The sensitivity of PCR often precludes the need for isolation and culture, thus making it ideal for the rapid detection of organisms involved in acute infections. We employed real-time (quantitative) PCR using TaqMan chemistry to detect leptospires in clinical and environmental samples. RESULTS AND CONCLUSIONS: The PCR assay can be applied to either blood or urine samples and does not rely on the isolation and culture of the organism. Capability exists for automation and high throughput testing in a clinical laboratory. It is specific for Leptospira and may discriminate pathogenic and non-pathogenic species. The limit of detection is as low as two cells.

Blood Specimen Collection↗
PubMed2007

Quantitative PCR based expression analysis on a nanoliter scale using polymer nano-well chips.

The analysis of gene expression is an essential element of functional genomics. Expression analysis is mainly based on DNA microarrays due to highly parallel readout and high throughput. Quantitative PCR (qPCR) based expression profiling is the gold standard for the precise monitoring of selected genes, and therefore used for validation of microarray data. Doing qPCR-based expression analysis in an array-like format can combine the higher sensitivity and accuracy of the qPCR methodology with a high data density at relatively low costs. This paper describes the development of an open-well based miniaturized platform for liquid PCR-based assays on the nanoliter scale using cost-effective polypropylene micro reactors (microPCR Chip). We show the quantification ability and reliability of qPCR in 200 nl with the microPCR chip down to 5 starting target molecules using TaqMan chemistry. An RNA expression analysis of four genes in mouse brain, liver and kidney tissues showed similar results in 200 nl as compared to standard 10 microl assays. The high sensitivity and quantification capability of the microPCR chip platform developed herein makes it a promising technology for performing high-throughput qPCR-based analysis in the nanoliter volume range.

Animals↗
PubMed1999

Quantitative PCR for counting residual white blood cells in blood products.

Leukocyte depleted blood components are frequently used to reduce alloimmunization and the risk of transfusion transmitted infection. Counting residual white blood cells in filtered blood products requires sensitive and reliable techniques. After separation of white blood cells from 500 microliters of 20 non-filtered and 54 filtered blood products we used polymerase chain reaction (PCR) and fluorimetric detection for the quantification of genomic DNA. The results were compared with results from Nageotte chamber counting. The accurate limit of detection of PCR was determined at 1 WBC/microliter (intra-assay coefficient of variation: 16.3%). PCR correlated well with Nageotte chamber counts (r = 0.77, p < 0.001, n = 74). Concordant results were obtained in 51 filtered and 20 non-filtered blood products. Discrepant results were obtained in 3 filtered whole blood units: In these blood products > 12 WBC/microliters were counted in Nageotte chamber and PCR gave a negative result. After component preparation fresh-frozen plasma and red cell concentrates of these units contained < 1 WBC/microliter using both methods. In conclusion we describe a quantitative PCR method which had about the same sensitivity and specificity as Nageotte chamber testing. However, PCR is more laborious than the standard method. As well, as reliable PCR testing requires expensive instruments and staff experienced in molecular biology, the standard method is more cost effective.

Fluorometry↗
PubMedDate not supplied

Acute parvovirus B19 infection in pregnant women--an analysis of serial samples by serological and semi-quantitative PCR techniques.

The serological and virological course of parvovirus B19 infection was followed in 14 women who suffered symptomatic or subclinical acute infection during pregnancy. Serial serum samples from the patients were tested for IgG and IgM antibodies and the levels of parvovirus B19 DNA were monitored using a semi-quantitative PCR assay. In addition, the outcome of the pregnancies was documented by clinical information and by testing cord blood for parvovirus B19 specific antibodies as well as for parvovirus B19 DNA by PCR. Levels of IgG antibodies rose steadily within 2 months of infection and in some cases began to decline at the end of pregnancy. IgM antibodies were usually detected for at least 2 months and persisted for as long as 9 months in one case. Viral DNA was detectable for at least 8 weeks following infection and semi-quantitative analysis revealed a gradual reduction in virus load during the viraemic phase of infection. There were no apparent differences in the course of antibody development and duration of viraemia in symptomatic versus subclinical infections.

Antibodies, Viral↗
PubMed2000

Stability in the HIV vDNA pool in peripheral CD4+ T cells of untreated patients by single tube quantitative PCR.

HIV infection leads to loss of CD4 T cells and development of AIDS in most individuals without treatment. While disease progression during HIV infection correlates with the plasma viral load, much less is known about the levels of HIV vDNA. This paper describes the development and validation of a sensitive, quantitative PCR assay for the assessment of HIV vDNA. The system uses novel single tube, multiply competitive PCR technology, which allows five-point competitor competition in a single PCR reaction. The reproducibility and performance characteristics of the assay are extensively studied, which indicate that the system performs well in high DNA backgrounds. Using this assay system on a cohort of protease naïve patients, HIV vDNA was assessed from PBMCs over an average follow-up period of 5 years. The data indicate that the HIV vDNA pool does not appreciably accumulate over the follow-up period, with many of the patients followed for up to 8 years. A reliable, quantitative assessment of vDNA pools will allow a better understanding of the dynamics of HIV pathogenesis.

CD4-Positive T-Lymphocytes↗
PubMed2006

Real-time quantitative PCR to detect changes in synovial gene expression in rheumatoid arthritis after corticosteroid treatment.

Synovial biomarkers are increasingly important in the development of novel therapeutic agents for the treatment of rheumatoid arthritis (RA). To identify biomarkers correlating with changes in clinical disease activity, real-time quantitative PCR (Q-PCR) was used to evaluate changes in synovial gene expression after treatment with corticosteroids. Patients with active RA received either oral prednisolone (n=10, 60 mg daily for the first week and 40 mg daily for the second week) or placebo (n=11) for 14 days. Real-time Q-PCR was used to quantify gene expression of tumour necrosis factor (TNF)alpha, IL1beta, IL8 and matrix metalloproteinase (MMP) 1 in synovial tissue samples obtained through an arthroscopic procedure before and after treatment. mRNA levels were reported as relative expression units compared with a cell-based standard. Statistical analysis was performed using an analysis of covariance model. Prednisolone markedly decreased IL8 and MMP1 expression compared with placebo, and the CIs excluded the likelihood of no effect. A trend towards reduction was seen in IL1beta and TNFalpha mRNA expression in the prednisolone group, although CIs included the value for no effect. These data suggest that Q-PCR can be used to measure synovial mRNA expression of mediators implicated in the pathogenesis of RA in small proof-of-concept trials.

Adult↗
PubMed2005

An improved method for determination of gene copy numbers in transgenic mice by serial dilution curves obtained by real-time quantitative PCR assay.

Precise characterization of transgene insertion is necessary for phenotype interpretation of transgenic animals. To check for the presence of deletions, estimate the number of inserted transgene copies, and in addition, identify the zygosity of transgenic mice, gene copy numbers were determined by real-time quantitative PCR. Instead of correlating tested samples to a single relative standard curve, serial dilution curves were constructed for every mouse sample. A novel statistical approach was designed in which mice with the same copy number were characterized by the adjusted group mean and standard deviation common to the target sequence. This enabled us to characterize the variability of the obtained results, statistically compare different groups of mice and estimate precision and limits of the applied method.

Analysis of Variance↗
PubMed2003

[Quantitative detection of hepatitis B virus by fluorescence quantitative PCR and its clinical value].

OBJECTIVE: To study the relationship between the serum contents of HBV DNA copies and the HBV serum index of immunology and the degree of hepatocyte injury in patients with HBV hepatitis. METHODS: The contents of HBV DNA copies were detected by fluorescence quantitative PCR (FQ-PCR), and the HBV serum index of immunology by ELISA. RESULTS: The contents of HBV DNA copies had a significant difference between HBeAg (+)/anti-HBe (-) group (42 patients) and HBeAg (-)/anti-HBe (+) group (57 patients) or HBeAg (-)/anti-HBe (-) group(11 patients) (P < 0.05), while there was no significant difference between HBeAg (-)/anti-HBe (+) group and HBeAg (-)/anti-HBe (-) group(P > 0.05). The contents of HBV DNA copies had no correlation with the severe degree of hepatocyte injury(P > 0.05). CONCLUSION: Anti-HBe is not the terminal sign of HBV replication. The severe degree of hepatocyte injury has not a direct correlation with the contents of HBV DNA copies.

Adolescent↗
PubMed1993

Electroconvulsive shock alters GABAA receptor subunit mRNAs: use of quantitative PCR methodology.

Electroconvulsive shock (ECS) may affect several neurotransmitter systems in brain, including the GABAergic inhibitory system. We used a quantitative PCR-based assay to evaluate mRNAs for five GABAa receptor subunits at 2 to 24 h after ECS. mRNAs for the alpha 1 and beta 2 subunits were significantly increased in cerebellum at 4 and 8 h after ECS, and returned to control levels at 24 h. No changes were observed in alpha 2, beta 3, gamma 1, or gamma 2 subunits, and no changes in any subunit evaluated were observed in cortex or hippocampus. These data corroborate prior results obtained for the alpha 1 subunit using Northern hybridization, and illustrate the utility of the PCR assay in quantitating low-abundance mRNAs.

Animals↗
PubMed2003

[Real-time quantitative PCR assay of Epstein-Barr virus in whole blood cells of nasopharyngeal carcinoma patients].

EBV is detected in more and more tumors, and is relative to carcinogenesis. We studied the copies of EBV DNA in whole blood of NPC patients and healthy controls by real-time quantitative PCR. In the 73 NPC patients and 83 controls, the positive rate of EBV in blood of NPC is 46.6%, while 13.3% in control. The mean copy number is 3.9 x 10(4) copys/microgram DNA in controls, which is much higher than NPC patients (which is 1.7 x 10(5) copies/microgram DNA). EBV infection is relative with NPC, while the lytic form of EBV maybe more important than its latent form. These results suggest that whole blood EBV DNA may be a valuable tool for molecular diagnosis of NPC.

Adult↗
PubMed2004

Expression of 25 kDa thiamine triphosphatase in rodent tissues using quantitative PCR and characterization of its mRNA.

Thiamine triphosphate (ThTP) is found in most organisms, but its biological role remains unclear. In mammalian tissues, cellular ThTP concentrations remain low, probably because of hydrolysis by a specific 25 kDa thiamine triphosphatase (ThTPase). The aim of the present study was to use quantitative PCR, for comparing the 25 kDa ThTPase mRNA expression in various mouse tissues with its enzyme activities. ThTPase mRNA was expressed at only a few copies per cell. The highest amount of mRNA was found in testis, followed by lung and muscle, while the highest enzyme activities were found in liver and kidney. The poor correlation between mRNA levels and enzyme activities might result either from tissue-specific post-transcriptional regulation of mRNA processing and/or translation or from the regulation of enzyme activities by post-translational mechanisms. Purified recombinant human ThTPase was phosphorylated by casein kinase II, but this phosphorylation did not modify the enzyme activity. However, the characterization of the 3'-untranslated mRNA region revealed a unique, highly conserved, 200-nucleotide sequence that might be involved in translational control. In situ hybridization studies in testis suggest a predominant localization of ThTPase mRNA in poorly differentiated spermatogenic cells. This is the first study demonstrating a cell-specific 25 kDa ThTPase mRNA expression, suggesting that this enzyme might be related to the degree of differentiation or the metabolic state of the cell.

3' Untranslated Regions↗
PubMed2003

Detecting homozygous deletions in the CDKN2A(p16(INK4a))/ARF(p14(ARF)) gene in urinary bladder cancer using real-time quantitative PCR.

PURPOSE: 9p21 is a major target in the pathogenesis of human urinary bladder cancer. The locus harbors the CDKN2A/ARF tumor suppressor gene, which encodes two cell cycle regulatory proteins cyclin dependent kinase 2A (p16(INK4a)) and alternate reading frame (p14(ARF)). We have designed a real-time quantitative PCR (QPCR) application to study homozygous deletion (HD) of CDKN2A/ARF in 186 urinary bladder cancer patients. EXPERIMENTAL DESIGN: Real-time QPCR, based on simultaneous amplification of ARF and a reference gene, GAPDH, was developed and evaluated in three melanoma cell lines with HDs at the CDKN2A/ARF locus (IGR-1, SK-MEL-5, and WM-266-4). In addition, loss of heterozygosity was analyzed at the D9S942, D9S1748, and D12S99 markers. Mutation analysis of the CDKN2A/ARF gene was performed using single-strand conformational polymorphism and sequencing. Results from the present investigation were combined with previous p53 analysis of the same urinary bladder neoplasms. RESULTS: Real-time QPCR analysis showed 26 (14%) HDs, 22 (12%) hemizygous deletions, and 3 (2%) multiple duplications. Loss of heterozygosity was determined in 30 (22%) cases at the D9S942 locus, which is located between E1alpha and E1beta of the CDKN2A/ARF gene. No association was established between occurrence of genetic aberrations at 9p21 and tumor stage or grade, supporting previous suggestions that CDKN2A/ARF inactivation is an early event in bladder carcinogenesis. CONCLUSIONS: We have established a fast and efficient method for detection of HDs. Our data support the notion that inactivation, including HDs, of CDKN2A/ARF is an early event in transitional cell carcinoma. We observed separate and specific targeting of the CDKN2A and ARF genes, respectively, and that simultaneous inactivation of ARF and p53 occurs.

Carcinoma, Transitional Cell↗
PubMed2006

Quantitative PCR confirms purity of strain GT, a novel trichloroethene-to-ethene-respiring Dehalococcoides isolate.

A novel Dehalococcoides isolate capable of metabolic trichloroethene (TCE)-to-ethene reductive dechlorination was obtained from contaminated aquifer material. Growth studies and 16S rRNA gene-targeted analyses suggested culture purity; however, the careful quantitative analysis of Dehalococcoides 16S rRNA gene and chloroethene reductive dehalogenase gene (i.e., vcrA, tceA, and bvcA) copy numbers revealed that the culture consisted of multiple, distinct Dehalococcoides organisms. Subsequent transfers, along with quantitative PCR monitoring, yielded isolate GT, possessing only vcrA. These findings suggest that commonly used qualitative 16S rRNA gene-based procedures are insufficient to verify purity of Dehalococcoides cultures. Phylogenetic analysis revealed that strain GT is affiliated with the Pinellas group of the Dehalococcoides cluster and shares 100% 16S rRNA gene sequence identity with two other Dehalococcoides isolates, strain FL2 and strain CBDB1. The new isolate is distinct, as it respires the priority pollutants TCE, cis-1,2-dichloroethene (cis-DCE), 1,1-dichloroethene (1,1-DCE), and vinyl chloride (VC), thereby producing innocuous ethene and inorganic chloride. Strain GT dechlorinated TCE, cis-DCE, 1,1-DCE, and VC to ethene at rates up to 40, 41, 62, and 127 micromol liter-1 day-1, respectively, but failed to dechlorinate PCE. Hydrogen was the required electron donor, which was depleted to a consumption threshold concentration of 0.76+/-0.13 nM with VC as the electron acceptor. In contrast to the known TCE dechlorinating isolates, strain GT dechlorinated TCE to ethene with very little formation of chlorinated intermediates, suggesting that this type of organism avoids the commonly observed accumulation of cis-DCE and VC during TCE-to-ethene dechlorination.

Chlorine↗
PubMed1998

Diagnosis of spinal muscular atrophy in an SMN non-deletion patient using a quantitative PCR screen and mutation analysis.

We report a child with clinical findings consistent with Werdnig-Hoffmann disease (spinal muscular atrophy type I) who was found not to have the homozygous absence of the survival motor neurone (SMN(T)) gene observed in approximately 95% of spinal muscular atrophy patients. A quantitative PCR based dosage assay for SMN(T) copy number showed that this patient possessed a single copy of the SMN(T) gene. Heteroduplex and sequence analysis of the remaining copy of SMN(T) showed a 2 base pair deletion within exon 4 which produces a frameshift and premature termination of the deduced SMN(T) protein. This protocol of initial SMN(T) gene dosage analysis followed by mutation detection allows identification of SMA compound heterozygotes (patients lacking one copy of SMN(T) and having another mutation in their other copy), thereby increasing the sensitivity of SMA molecular diagnosis.

Cyclic AMP Response Element-Binding Protein↗
PubMed2000

Retinal VEGF mRNA measured by SYBR green I fluorescence: A versatile approach to quantitative PCR.

PURPOSE: To determine whether continuous monitoring of SYBR Green I fluorescence provides a reliable and flexible method of quantitative RT-PCR. Our aims were (i) to test whether SYBR Green I analysis could quantify a wide range of known VEGF template concentrations, (ii) to apply this method in an experimental model, and (iii) to determine whether 20 existing primer pairs could be used to quantify their cognate mRNAs. METHODS: Real-time quantitative PCR was performed using a LightCycler rapid thermal cycler (Roche). Retinal VEGF mRNA levels were measured in a murine model of oxygen-induced retinopathy during vaso-obliterative and hypoxic phases. RESULTS: This technique was able to detect as few as 10 control template copies, with quantitative data available routinely for 1000 or more copies. The levels of retinal VEGF mRNA expression followed the hypoxia-induced pattern determined previously by conventional methods. All gene-specific primer pairs which amplify a specific product by conventional PCR were successfully converted to SYBR Green analysis, including those for housekeeping genes glyceraldehyde phosphate dehydrogenase (GAPDH), cyclophilin, and acidic ribosomal phosphoprotein PO (ARP/36B4) and for 28S rRNA. In each case melting curve analysis and agarose gel electrophoresis confirmed the specificity of the amplification product. CONCLUSIONS: The sequence-independent detection of DNA with SYBR Green I means that it can be used to quantify the amplification of any cDNA using gene-specific primers. This rapid and flexible method is ideally suited for researchers in vision science wishing to quantify mRNAs from many different genes because it does not require investment in gene-specific hybridization probes.

Animals↗