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Results for “Quantitative PCR”

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At least 1,207 records · Page 67Linked to original sources
PubMed2002

Her-2/neu expression and gene amplification in gastrinomas: correlations with tumor biology, growth, and aggressiveness.

A proportion of gastrointestinal neuroendocrine tumors are aggressive; however, little is known of molecular determinants of their growth, and molecular studies have identified no useful prognostic factors. Overexpression of HER-2/neu is common in some nonendocrine tumors, frequently correlates with increased tumor aggressiveness, and can be used as a basis of treatment with trastuzumab. Little is known of its expression in malignant pancreatic endocrine tumors. In the present study HER-2/neu gene amplification and expression was determined in 43 gastrinomas from different patients. Results were correlated with clinical, laboratory, and tumor characteristics including tumor growth. HER-2/neu gene amplification was assessed by differential PCR, mRNA levels assessed by quantitative PCR, and protein by immunohistochemistry. Fourteen percent of patients had HER-2/neu gene amplification in tumors compared with levels in their WBCs. HER-2/neu mRNA varied over a 700-fold range. However, only 3% exceeded levels seen in normal pancreas, and immunohistochemistry did not show protein overexpression in any tumor (n = 10). HER-2/neu mRNA levels were significantly higher (P = 0.032) in tumors associated with liver metastases but not with tumor location or size. These results show that HER-2/neu amplification/overexpression does not seem to play a role in the molecular pathogenesis of most gastrinomas, as suggested in a previous study involving small numbers of cases. However, mild gene amplification occurs in a subset, and overexpression is associated with aggressiveness. Therefore, HER-2/neu levels could have prognostic significance as well as identify a patient subset with gastrinomas who might benefit from trastuzumab treatment.

Adolescent↗
PubMed2002

Role of DNA methylation in transcription of human endogenous retrovirus in the pathogenesis of systemic lupus erythematosus.

OBJECTIVE: We recently reported that transcription of human endogenous retrovirus (HERV) clone 4-1-like sequences is increased in patients with systemic lupus erythematosus (SLE). We therefore investigated the role of DNA methylation in the transcription of this HERV. METHODS: The effect of a demethylating agent, 5-aza-deoxycytidine (5-aza C), on the transcription of HERV clone 4-1 in healthy individuals and patients with SLE was examined using reverse transcriptase-PCR and real-time quantitative PCR. RESULTS: 5-aza C increased clone 4-1-like messenger RNA in healthy controls, but not in patients with SLE. CONCLUSION: Defects of methylation may contribute to the transcription of HERV in patients with SLE and this may be related to the pathogenesis of SLE.

Adult↗
PubMed2005

Gene expression profiles of intact and regenerating zebrafish retina.

PURPOSE: Investigate the molecular determinants of retinal regeneration in adult vertebrates by analyzing the gene expression of control and post-lesion retina of adult zebrafish, a system that regenerates following injury. METHODS: Gene expression of zebrafish retina and brain were determined with DNA microarray, RT-PCR, and real-time quantitative PCR analyses. Damaged retinas and their corresponding controls were analyzed 2-5 days post-lesion (acute injury condition) or 14 d post-lesion (cell regeneration condition). RESULTS: Expected similarities and differences in the gene expression profile of zebrafish retina and brain were observed, confirming the applicability of the gene expression techniques. Mechanical lesion of retina triggered significant, time-dependent changes in retinal gene expression. The induced transcriptional changes were consistent with cellular phenomena known to occur, in a time-dependent manner, subsequent to retinal lesion, including cell cycle progression, axonal regeneration, and regenerative cytogenesis. CONCLUSIONS: The results indicate that retinal regeneration in adult zebrafish involves a complex set of induced, targeted changes in gene transcription, and suggest that these molecular changes underlie the ability of the adult vertebrate retina to regenerate.

Animals↗
PubMed2006

[Expression of CXCR3 in hepatocellular carcinoma].

BACKGROUND & OBJECTIVE: Recent researches have revealed that chemokine-chemokine receptor network plays an important role in tumor metastasis. This study was to investigate the expression of CXCR3, a chemokine receptor, and its correlation to clinicopathologic features of hepatocellular carcinoma (HCC). METHODS: CXCR3 mRNA expression in 7 human HCC cell lines, 18 normal liver tissues and 64 pairs of HCC tissues and adjacent liver tissues was detected by reverse transcription-polymerase chain reaction (RT-PCR) and real-time quantitative PCR; CXCR3 protein expression was detected by immunohistochemistry. The correlation of CXCR3 expression to the metastasis of HCC was analyzed. RESULTS: CXCR3 mRNA was expressed in 2 HCC cell lines (MHCC97-H and MHCC97-L) with metastatic potential, but not in 5 HCC cell lines without metastatic potential. The CXCR3/beta2M expression ratio in MHCC97-H cells (high metastatic potential) was 33.0x10(-4), and that in MHCC97-L cells (low metastatic potential) was 8.7x10(-4). Strong staining of CXCR3 protein in MHCC97-H and MHCC97-L cells was observed. However, the staining of CXCR3 protein was not detected in 4 HCC cell lines without metastatic potential; only weak staining was detected in HepG-2 cells. The expression of CXCR3 protein in HCC is significantly correlated to the invasiveness of tumors (P=0.003). The 1-and 2-year tumor-free survival rates of the HCC patients with strong positive CXCR3 expression were significantly lower than those of the patients with weak positive CXCR3 expression (66.7% vs. 75.0%, 31.3% vs. 59.5%, P=0.044). CONCLUSION: The expression of CXCR3 might play a role in the invasion and metastasis of HCC.

Adult↗
PubMedDate not supplied

[Molecular biology at the service of the daily medical virology. 1. Methodological principles].

Molecular biology techniques which can rapidly detect a few copy number of viral genome allow to obtain new parameters for the diagnosis, the prognosis, and the monitoring of viral diseases in addition to or in substitution for cell culture and antigen and antibody detection. The methods are based on the virus structural organization and on their replication into cells. They use the tools which have been developed to analyse nucleic acids. Molecular hybridization (dot-blot, southern-blot, liquid phase hybridization, in situ hybridization), enzymatic amplification (PCR, nested-PCR, RT-PCR, NASBA, bDNA, multiplex PCR), DNA and RNA quantification (quantitative PCR or RT-PCR, NASBA, bDNA) and genotyping (virus types, mutations, variant virus) are used. Nucleic acid extracts native or amplified with a reporter molecule (dUTP-biotin or dUTP-digoxigenin) are identified using gel electrophoresis followed by hybridization or using microwell capture hybridization assay and colorimetric immunoenzymatic or bioluminescence assays.

Genetic Engineering↗
PubMed2004

[Inhibition of hepatitis B virus replication by RNA interference in vitro].

OBJECTIVE: To design pSilencer3.1-H1hygro plasmid expressing short interfering RNAs (siRNA) that targets HBV core gene region, and to evaluate inhibitory effect of this siRNA on HBV in vitro. METHODS: HepG2 2.2.15 was used as target cells. The plasmid and liposome metafectene were cotransfected into the cultured cells, HBV DNA were analyzed by fluorogenic quantitative PCR (FQ-PCR), HBV C-mRNA was detected by semi-quantitative RT-PCR. RESULTS: The plasmid expressing siRNA was successfully constructed. The two constructed siRNAs could effectively inhibit HBV replication, and their inhibitive effect on HBV was dose-dependent. CONCLUSION: These results showed that siRNA could substantially inhibit HBV replication in the infected cells

Hepatitis B virus↗
PubMed2005

[Inhibition of hepatitis B virus replication and expression by RNA interference in vitro].

OBJECTIVE: To design pSilencer3.1-H1hygro plasmid expressing short interfering RNAs (siRNA) that target HBV S gene region, and to evaluate inhibitory effect of this siRNA on HBV in vitro. METHODS: HepG2.2.15 was used as target cell. The plasmid expressing small interfering RNA was transfected into the cultured cells via liposome metafectene, HBsAg and HBeAg were analyzed by time-resolved immunofluorometric assay, HBV DNA were analyzed by fluorogenic quantitative PCR (FQ-PCR), HBV S-mRNA was detected by semi-quantitative RT-PCR. RESULTS: The plasmid expressing siRNA was successfully constructed. The S region siRNAs could effectively inhibit both antigens secretion and HBV replication compared with controls, HBsAg levels decreased by 75%, 82%, 89%; HBeAg levels decreased by 32%, 38%, 43%; HBV DNA production decreased by 30%, 43%, 49%; The HBV mRNA species was reduced by 30%, 70%, 90% when transfected with 1 microg, 2 microg, 4 microg HBV S-siRNA, respectively. CONCLUSION: These results demonstrate that RNAi can substantially inhibit HBV replication and the antigens expression in the infected cells. These inhibitive effect of siRNA on HBV was dose-dependent and sequence-specific.

Gene Expression Regulation, Viral↗
PubMed2006

[Inhibition of hepatitis B virus replication and expression by RNA interference in vivo].

OBJECTIVE: To evaluate the inhibitory effect of small interfering RNA (siRNA) targeting HBV C gene region on hepatitis B virus (HBV) in vivo. METHODS: An animal model of HBV infection was developed hydrodynamically, and pcDNA3.1-HBV and siRNA were together injected into the tail vein of the BALB/c mice. HBsAg was analyzed by time-resolved immunofluorometric assay, HBV DNA was analyzed by fluorogenic quantitative PCR (FQ-PCR), HBV C-mRNA was detected by semi-quantitative RT-PCR, and viral specific proteins (HBsAg and HBcAg) in the mice livers were assayed using immunohistochemical staining. RESULTS: In the mice, the siRNA effectively inhibited HBV replication and expression compared with the controls. The inhibitive effect of siRNA on HBV lasted at least 3 days. CONCLUSION: These results demonstrate that RNAi can substantially inhibit HBV replication and expression in vivo.

Animals↗
PubMed1994

Measurement of tissue factor messenger RNA levels in human endothelial cells by a quantitative RT-PCR assay.

We have developed a sensitive and quantitative RT-PCR assay for the determination of tissue factor (TF) mRNA levels in human cells. An in vitro synthesized internal standard RNA was used to correct for differences in reverse transcription or amplification of various RNA samples. The PCR products were quantitated by hybridization. The sensitivity was such that less than 0.2 microgram of total endothelial RNA sufficed to measure its TF mRNA content. The RT-PCR assay was used to determine TF mRNA levels in endothelial cells treated with a factor from human melanoma cells and/or TNF. In this way the amount of TF mRNA could be induced to a level that was at least 80-fold higher than that in non-induced cells. This increase was in the same order of magnitude as the induction of measured TF activity.

Base Sequence↗
PubMed2004

Instant evaluation of the absolute initial number of cDNA copies from a single real-time PCR curve.

Amplification of a cDNA product by quantitative PCR (qPCR) is monitored by a fluorescent signal proportional to the amount of produced amplicon. The qPCR amplification curve usually displays an exponential phase followed by a non-exponential phase, ending with a plateau. Contrary to prevalent interpretation, we demonstrate that under standard qPCR conditions, the plateau can be explained by depletion of the probe through Taq polymerase- catalysed hydrolysis. Knowing the probe concentration and the fluorescence measured at the plateau, a specific fluorescence can thus be calculated. As far as probe hydrolysis quantitatively reflects amplicon synthesis, this, in turn, makes it possible to convert measured fluorescence levels in the exponential phase into concentrations of produced amplicon. It follows that the absolute target cDNA concentration initially engaged in the qPCR can be directly estimated from the fluorescence data, with no need to refer to any calibration with known concentrations of target DNA.

Animals↗
PubMed1992

Regulation of aromatase cytochrome P-450 (estrogen synthetase) transcripts in the quail brain by testosterone.

The aromatase cytochrome P-450 (P-450AROM) cDNA, which was identified by homologies in the DNA and in the deduced amino acid sequences with human P-450AROM cDNA, was isolated from a brain cDNA library of Japanese quail, demonstrating the presence of RNA transcripts of P-450AROM in the quail brain. To determine trace amounts of P-450AROM mRNA in the brain and to examine the effects of testosterone on its expression, a quantitative PCR method of RNA transcripts was developed. Brain total RNA was subjected to reverse transcription reaction and then quantitated by PCR from cDNA with a fluorescent dye-labeled primer. The quantity of P-450AROM mRNA was calculated by using an internal standard of modified P-450AROM (m-P-450AROM) RNA. The brain P-450AROM was primarily transcribed in the hypothalamus area (1.15 +/- 0.14 amol/micrograms of RNA) and traces of transcripts only were detected in the cerebellum (0.038 +/- 0.005 amol/micrograms of RNA). The P-450AROM mRNA in the hypothalamus of castrated quail was low (0.270 +/- 0.078 amol/micrograms of RNA) and increased 4- to 5-fold following treatment with testosterone. These results demonstrate, for the first time, that the increase in P-450AROM activity that is observed in the brain following treatment with testosterone results from a pretranslational regulation of the P-450AROM by androgens.

Androgens↗
PubMed2003

Measurement of human cytomegalovirus loads by quantitative real-time PCR for monitoring clinical intervention in transplant recipients.

Quantitative monitoring of human cytomegalovirus (HCMV) infection is helpful in determining appropriate antiviral management of transplant recipients. Quantitative PCR technologies have demonstrated accuracy in measuring systemic HCMV loads. A total of 298 consecutive whole-blood specimens submitted to the Clinical Virology Laboratory at Vanderbilt University Medical Center from 15 February to 31 October 1999 were included in the study. In addition to a qualitative colorimetric microtiter plate PCR assay (MTP-PCR) and a semiquantitative pp65 antigenemia assay, each specimen was measured for HCMV loads by a quantitative PCR assay performed on an ABI PRISM 7700 Sequence Detection System (TaqMan). Compared to results of the MTP-PCR, the sensitivity, specificity, positive predictive value, and negative predictive value were 70.5, 97.5, 87.8, and 92.8% for the antigenemia assay and were 96.7, 92.0, 75.6, and 99.1% for the TaqMan assay, respectively. There was a high correlation between antigenemia values and HCMV loads as determined by the TaqMan (r = 0.989; P < 0.001). Antigenemia values of 0, 1 to 10, 11 to 100, 101 to 1,000, and over 1,000 positive cells per 2 x 10(5) leukocytes corresponded to median HCMV loads measured by TaqMan of 125, 1,593, 5,713, 16,825, and 5,425,000 copies/ml, respectively. Corresponding to antigenemia values of 1 to 2, 10, and 50 positive cells per 2 x 10(5) leukocytes, HCMV viral loads of 1,000, 4,000, and 10,000 copies/ml are proposed as cutoff points for initiating antiviral therapy in patient groups with high, intermediate, and low risk of CMV diseases.

Cytomegalovirus↗
PubMed2004

Comparison of two standardisation methods in real-time quantitative RT-PCR to follow Staphylococcus aureus genes expression during in vitro growth.

By real-time quantitative PCR (RTQ-PCR), two different standardisation methods were used to quantify expression of three target genes (RNAII and RNAIII transcripts of agr locus and ica transcript of icaADBC locus): (i) a relative quantification, using a transcript of three housekeeping genes (gyrase A, gyrA; guanylate kinase, gmk and 16S rRNA, 16S) as internal standard, and (ii) an absolute quantification, using cloned sequences of the target genes in known concentrations as external standards. To determine the efficiency and reliability of these two methods, the gene expressions were studied during the growth of a clinical isolate of Staphylococcus aureus. Between 3 and 20 h after inoculation, target gene transcription was analysed using LightCycler Apparatus, LC Data Analysis software and RelQuant software for relative quantification (Roche). For all target genes, the expression profiles obtained with gyrA or gmk as internal standards remained almost identical. However, these profiles varied between each other depending on the standard gene. Due to their important expression variations during growth phases, these two housekeeping genes seem inappropriate to be used as internal standards. The absolute quantification of the three transcripts of interest gave results similar to their relative quantification expressed versus 16S rRNA. Therefore, our study suggests the suitable use of 16S rRNA as internal standard in RTQ-PCR quantification of staphylococcal gene expression during the stationary phase of growth.

DNA Gyrase↗
PubMed2004

Allele-frequency determination of BsmI and FokI polymorphisms of the VDR gene by quantitative real-time PCR (QRT-PCR) in pooled genomic DNA samples.

The vdr gene is a candidate for osteoporosis susceptibility, with conflicting results in association studies. We have designed and optimized an individual allele-specific and DNA pooling PCR-based methodology to quantitate BsmI and FokI polymorphisms of the vdr gene and studied single-nucleotide polymorphisms (SNPs) from pooled DNA samples. The allele frequency in DNA pooling experiments has been analyzed by kinetic PCR: quantitative real-time PCR (QRT-PCR). A Spanish cohort of 225 healthy postmenopausal women was studied. Bone mineral density (BMD) was measured by dual-energy X-ray absorptiometry (DEXA) and quantitative ultrasound calcaneous densitometry. Allele-specific PCR amplification of BsmI and FokI genes showed full concordance with the PCR-RFLP approach. The prevalence of the three BsmI VDR genotypes was 19.1, 44.9 and 36.0% for BB, Bb and bb, respectively. In the case of the FokI locus, the prevalence of genotypes was 40.4, 48.0 and 11.6% for FF, Ff and ff, respectively. No positive correlation was found between polymorphism and BMD. The DNA pooling procedure was validated. No differences were found in allele frequencies and T-score data obtained using the high throughput DNA pooling approach, as compared to known individual frequencies. In our hands, this is a very useful approach to study quantitative (thus polygenic) traits like osteoporosis susceptibility.

Absorptiometry, Photon↗
PubMed1995

Quantitative RT-PCR for measuring gene expression.

Classical Northern blot analysis for measuring mRNA requires too many cells to be practical for cell sorting. Yet, measurement of gene expression in small subsets within a heterogeneous population of cells is often desired. The PCR in combination with prior reverse transcription (RT-PCR) of the mRNA of interest provides a means for measuring gene expression using as few as one cell. When RT-PCR is performed, the reliability of the data can be highly subjective due to the efficiency of both RT and PCR steps. This subjectivity can be eliminated by a technique for quantitating specific RNA molecules using an internal RNA competitive reference standard (RNA-CRS), which is identical to the sequence of interest except for a deletion of 80 bases. Here we illustrate a strategy for quantitative PCR using a RNA-CRS, synthesized solely using nonplasmid-based PCR techniques. The competitive reaction consists of a constant quantity of wild-type mRNA (from 100-1000 cells) added individually to tubes containing a serially decreasing amount of RNA-CRS. The RT-PCR is performed on these samples, then the resulting product is analyzed by gel electrophoresis and densitometry. The procedure for preparing the RNA-CRS and subsequent RT-PCR steps are described in detail.

Animals↗
PubMed2002

Quantification of human mitochondrial DNA in a real time PCR.

Recently, a moderately priced machine for real-time quantitative PCR has become available, the Perkin Elmer 5700. The rapid and quantitative assay of mitochondrial DNA (mtDNA) copy number is potentially useful in a variety of molecular, evolutionary and forensic fields. Using this new tool, we have evaluated the precision and reliability of the real time PCR to quantify undeleted mitochondrial genome copy number, and to determine the frequency of an age-associated deletion of 4977 base pairs in length, in 42 human iliopsoas muscle DNA samples from persons of known age. We have evaluated the accuracy with which age can be predicted, knowing only the frequency of this common 4977 bp deletion, and derived a statistical formula which describes the confidence with which the 4977 bp frequency predicts age. The results indicate that the mutation frequency could be used to distinguish between tissue from young and old individuals. However in this data set, while there was considerable agreement of 4977 bp frequency among replicates from the same individual sample, there was substantial diversity of mean mutation frequency between individuals of the same or similar ages. The simplest interpretation of these results is that there are biological modifiers of 4977 bp frequency that are age-independent, which are potentially interesting but may limit the usefulness of this deletion frequency alone as a "molecular forensic clock."

Autopsy↗
PubMed2004

Real-time PCR-based gene dosage assay for detecting BRCA1 rearrangements in breast-ovarian cancer families.

BRCA1 and BRCA2 germline mutations, mainly point mutations and other small alterations, are responsible for most hereditary cases of breast-ovarian cancer. However, the observed frequency of BRCA1 alterations is lower than that predicted by linkage analysis. Several large BRCA1 rearrangements have been identified with a variety of technical approaches in some families. We have developed a gene dosage assay based on real-time quantitative PCR and used it to extensively analyze 91 French families of breast-ovarian cancer in which no BRCA1 or BRCA2 point mutations was identified. This gene dosage method calculates the copy number of each BRCA1 exon to readily detect one, two, and three or more copies of BRCA1 target exons. In the series of 91 families at high risk of carrying BRCA1 mutations, we detected seven large rearrangements of the BRCA1 gene by using this real-time PCR approach. This simple, rapid, and semiautomated real-time quantitative polymerase chain reaction (PCR) assay is a promising alternative technique to Southern blot, bar code analysis on combed DNA, quantitative multiplex PCR of short fluorescent fragments, and cDNA length analysis for the detection of large rearrangements. Therefore, this technique should be considered as a powerful diagnostic method for breast/ovarian cancer susceptibility in clinical and research genetic surveys.

Base Sequence↗