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At least 775 records · Page 43Linked to original sources
PubMed2004

High-level expression of the coxsackievirus and adenovirus receptor messenger RNA in osteosarcoma, Ewing's sarcoma, and benign neurogenic tumors among musculoskeletal tumors.

PURPOSE: The sensitivity of human tumor tissues to infection with recombinant adenoviruses correlates with the expression of the coxsackievirus and adenovirus receptor (CAR). CAR has been shown to function as the primary receptor for adenoviruses and to play a critical role in adenovirus entry into host cells. It is important for clinical gene therapy to determine the expression level of CAR in tumor tissues. EXPERIMENTAL DESIGN: We analyzed the expression of CAR mRNA in 154 musculoskeletal tumor tissues from 154 patients and 10 normal mesenchymal tissues from 3 patients using reverse transcription-PCR and real-time quantitative PCR. An adenovirus infection assay was performed in two cell lines that were established from CAR-positive osteosarcoma tissue and CAR-negative malignant fibrous histiocytoma tissue. RESULTS: Ninety-nine of 154 tumors were detected as CAR positive by reverse transcription-PCR. We found that the expression levels of CAR mRNA varied markedly between different tumors as determined by real-time quantitative PCR. CAR mRNA was expressed at high levels in osteosarcoma, Ewing's sarcoma, neurofibroma, and schwannoma; at intermediate levels in exostosis, giant cell tumor, liposarcoma, synovial sarcoma, malignant peripheral nerve sheath tumor, and hemangioma; and at low levels in alveolar soft part sarcoma and desmoid. Whereas the osteosarcoma cell line that expressed a high level of CAR mRNA, like its parent tumor, had a high efficiency of adenovirus infection, the malignant fibrous histiocytoma cell line with almost undetectable expression of CAR mRNA, like its parent tumor, had a low efficiency of infection. CONCLUSIONS: Our data showed the great variations in CAR mRNA expression among human musculoskeletal tumors and mesenchymal tissues and implicated the potential usefulness of adenoviral vectors in gene therapy for osteosarcoma, Ewing's sarcoma, neurofibroma, and schwannoma. Efficient transduction with adenovirus for gene therapy could be realized in appropriate, sensitive tumor types.

Adenoviridae↗
PubMed2006

Diagnosis and monitoring of human cytomegalovirus infection in bone marrow transplant recipients by quantitative competitive PCR.

OBJECTIVES: Human cytomegalovirus (HCMV) is a common cause of infection worldwide. Severe cytomegalovirus disease is usually observed in immunodeficient individuals such as bone marrow transplant (BMT) or AIDS patients. In these patients, proof of viral presence is not enough for making clinical decisions; one must report the quantity of virus or viral load in appropriate clinical specimens to demonstrate the relationship between disease severity and HCMV infection. The goal of this study was to use quantitative competitive polymerase chain reaction (PCR) to determine HCMV viral load in 26 BMT recipients. MATERIALS AND METHODS: Peripheral blood was collected weekly for 100 days from 26 BMT recipients. Qualitative and quantitative competitive PCRs on 10(5) mononuclear cells were performed for each patient. The same tests were performed once for each of 26 donors. In addition, the anti-HCMV humoral response was detected by performing IgM and IgG ELISAs in donors and recipients prior to transplantation. RESULTS: Of 26 BMT donors and recipients, 25 and 26 were IgG positive, and 2 and 6 had HCMVspecific IgM antibodies, respectively. From 313 total clinical specimens tested, 255 had positive qualitative PCR results. Results of quantitative PCR on the same specimens demonstrated that in 14 patients, viral copy number per 10(5) cells had increased, pointing toward HCMV reactivation. In others, changes in viral copy number were mostly around 100/10(5) cells, with an upper limit of 300/10(5) cells. CONCLUSIONS: Owing to the high prevalence of cytomegalovirus in our country, the chance of viral reactivation and HCMV infection/disease upon transplantation must be seriously considered. Therefore, use of quantitative PCR in PCR-positive patients is highly recommended to demonstrate active infection that may lead to HCMV disease during the posttransplant period. This also could help physicians begin pre-emptive therapy that would be for a shorter treatment period and provide for better outcomes in infected BMT patients.

Adolescent↗
PubMed1993

Competitive PCR for quantitation of gonadotropin-releasing hormone mRNA level in a single micropunch of the rat preoptic area.

A competitive polymerase chain reaction (PCR) for quantitating gonadotropin-releasing hormone (GnRH) mRNA level in a single micropunch of the rat preoptic area (POA) is described. The POA (600 microns in depth) was micropunched from frozen rat brain slices and used for mRNA isolation using Dynabeads-oligo(dT) magnetic separation technique. The target RNA combined with a synthetic, deletion mutant GnRH cRNA as an internal standard, is co-reverse transcribed, and their cDNAs are subsequently co-amplified by Taq DNA polymerase in the same tube in which the same GnRH primers are used. This PCR protocol is sensitive enough to detect GnRH mRNA level in a single POA micropunch derived from an individual rat. There is a linear increase of the amount of GnRH PCR products as a function of input RNA and of the number of PCR cycles. Addition of mutant GnRH cRNA as an internal standard allows us to quantitate GnRH mRNA level in biological samples and to compensate variations of PCR reaction between samples. Following preoptic treatment with 5'-ADMP, which depletes selectively norepinephrine (NE), GnRH mRNA level was significantly reduced. This simple, yet highly sensitive PCR method appears to be a valuable tool for the study of the cellular and molecular regulation of GnRH gene expression in a variety of experimental models.

Animals↗
PubMed2006

Multiplex ligation-dependent probe amplification for rapid detection of proteolipid protein 1 gene duplications and deletions in affected males and carrier females with Pelizaeus-Merzbacher disease.

BACKGROUND: Pelizaeus-Merzbacher disease is a rare X-linked neurodegenerative disorder caused by sequence variations in the proteolipid protein 1 gene (PLP1). PLP1 gene duplications account for approximately 50%-75% of cases and point variations for approximately 15%-20% of cases; deletions and insertions occur infrequently. We used multiplex ligation-dependent probe amplification (MLPA) to detect PLP1 gene alterations, especially gene duplications and deletions. METHODS: We performed MLPA on 102 samples from individuals with diverse PLP1 gene abnormalities and from controls, including 50 samples previously characterized for the PLP1 gene by quantitative PCR but which were anonymized for prior results and sex. RESULTS: All males with PLP1 gene duplications (n = 13), 1 male with a triplication, 2 males with whole gene deletions, and all controls (n = 72) were unambiguously assigned to their correct genotype. Of 4 female carriers tested by MLPA and quantitative PCR, 3 were duplication carriers by both methods, and 1 was a triplication carrier by MLPA and a duplication carrier by quantitative PCR. For 1 sample with a partial deletion, MLPA showed exon 3 deleted but PCR showed exons 3 and 4 deleted. Sequence analysis of 2 samples with reduced dosage for exons 3 and 5 revealed point variations overlapping the annealing site for the corresponding MLPA probe. The precision of MLPA analysis was excellent and comparable to or better than quantitative PCR, with CVs of 4.3%-9.8%. CONCLUSIONS: MLPA is a rapid and reliable method to determine PLP1 gene copies. Samples with partial PLP1 gene dosage alterations require confirmation with a non-MLPA method.

Carrier State↗
PubMed2003

Growth of the postnatal rat retina in vitro: quantitative RT-PCR analyses of mRNA expression for photoreceptor proteins.

PURPOSE: To investigate whether previously reported changes in protein expression of middle and long (M/L) and short (S) wavelength cone opsin pigments in cultured retina are correlated with changes in their gene expression. Additionally, to elucidate the importance of a functional retinal pigment epithelium for the development of photoreceptor outer segments. METHODS: Neonatal rat retinas were maintained in culture for 11 days and either fixed in 4% paraformaldehyde for immunohistochemistry or prepared for RNA extraction, reverse transcription polymerase chain reaction (RT-PCR), and quantitative RT-PCR. S-cone and M/L-cone photoreceptors as well as rod photoreceptors were immunohistochemically identified using specific antibodies. Peanut agglutinin (PNA)-lectin histochemistry was used to identify interphotoreceptor matrix associated with cone photoreceptors. Immunolabeling for ED-1 and RPE65 was performed in combination with PNA-lectin staining to examine interactions between photoreceptor cells and the retinal pigment epithelium. Relative estimates of mRNA expression levels for M/L-opsin, S-opsin, recoverin, and rhodopsin in normal and cultured retina were determined by using quantitative RT-PCR. RESULTS: Strong immunolabeling for recoverin and rhodopsin accumulated in outer segments as well as photoreceptor somata in vitro. Cultured and normal retinas showed similar relative expression levels of recoverin and rhodopsin mRNA. In cultured rat retina, the density of S-cones was high and M/L-cones could not be immunohistochemically detected. However, M/L-cone photoreceptor mRNA was detectable, but at a fourfold lower level in cultured than in vivo retinas. The S-cone photoreceptor mRNA level was almost twofold lower than in vivo. Retinal pigment epithelium cells in cultured specimens showed no RPE65 immunolabeling, but expressed immunolabeling for ED-1 indicating phagocytic activity of these cells in vitro. CONCLUSIONS: We assume that the high density of S-cones and virtually no M/L-cones seen in in vitro retinas might represent an immature stage with numerous S-cones and suppressed transdifferentiation into M/L-cone phenotype. A non-functional relationship between photoreceptor cells and a dysfunctional retinal pigment epithelium may have severe consequences for the development of outer segments.

Animals↗
PubMed2003

Screening for large rearrangements of the BRCA1 gene in German breast or ovarian cancer families using semi-quantitative multiplex PCR method.

Since the identification of the breast and ovarian cancer susceptibility genes BRCA1 and BRCA2, a large number of different germline mutations in both genes have been found by conventional PCR-based mutation detection methods. Complex germline rearrangements such as those reported in the BRCA1 gene are often not detectable by these standard diagnostic techniques. To detect large deletions or duplications encompassing one or more exons of the BRCA1 gene and in order to estimate the frequency of BRCA1 rearrangements in German breast or ovarian cancer families, a semi-quantitative multiplex PCR method was developed and applied to DNA samples of patients from families negatively tested for disease causing mutations in the BRCA1 and BRCA2 coding regions by direct sequencing. Out of 59 families analysed, one family was found to carry a rearrangement in the BRCA1 gene (duplication of exon 13). The results indicate that the semi-quantitative multiplex PCR method is useful for the detection of large rearrangements in the BRCA1 gene and therefore represents an additional valuable tool for mutation analysis of BRCA1 and BRCA2.

Adult↗
PubMed2004

E-cadherin mutation and Snail overexpression as alternative mechanisms of E-cadherin inactivation in synovial sarcoma.

We have recently reported frequent E-cadherin gene mutations in synovial sarcoma (SS), suggesting mutational inactivation of E-cadherin as a potential mechanism of spindle cell morphology in SS, a spindle cell sarcoma that shows areas of glandular epithelial differentiaton in some cases (biphasic SS) and only pure spindle cell morphology in most cases (monophasic SS). However, the mechanism of downregulation of E-cadherin in SS remains unknown. To further address this issue, we analysed the mechanisms of E-cadherin silencing in 40 SS. Genetic and epigenetic changes in the E-cadherin gene, and the expression level of its transcriptional repressor Snail were examined by polymerase chain reaction-single-strand conformation polymorphism (PCR-SSCP), methylation-specific PCR, and real-time quantitative PCR, respectively. Expression of E-cadherin was examined by RT-PCR and immunohistochemistry. We also examined ELF3, a transcription factor associated with epithelial differentiation in SS in a previous cDNA microarray, by RT-PCR. E-cadherin and ELF3 transcripts were detected, respectively, in 27/40 (67.5%) and in 25/40 (62.5%) of SS, and these epithelial-related genes were almost always coexpressed. Hypermethylation of the promoter of the E-cadherin gene was detected in five cases (12.5%) in SS; however, E-cadherin was silenced at mRNA level in only one of the five cases. E-cadherin missense mutations were observed in five cases (12.5%) of SS. In SS, all five cases with E-cadherin missense mutations had the SYT-SSX1 fusion and were monophasic tumors, suggesting a relationship between the SYT-SSX fusion type and E-cadherin missense mutation (P=0.07). E-cadherin mRNA expression in SS was associated with reduced Snail expression level (P=0.03). E-cadherin membranous expression was observed in 14/40 (35.0%) of SS, and was also correlated with SYT-SSX1 fusion type and biphasic histology. ELF3 was confirmed to be more highly expressed in biphasic than monophasic SS by real-time quantitative PCR. These results suggest that in SS the loss of E-cadherin expression occurs either by Snail trans-repression or by inactivating mutations. Thus, E-cadherin downregulation is associated with the loss or absence of glandular epithelial differentiation in certain SS.

Cadherins↗
PubMed2006

Development of a quantitative-competitive PCR for quantification of human cytomegalovirus load and comparison with antigenaemia, viraemia and pp67 RNA detection by nucleic acid sequence-based amplification.

AIM: The human cytomegalovirus (HCMV) is an important pathogen in immunocompromised patients, such as transplant recipients. The use of sensitive and rapid diagnostic assays can have a great impact on antiviral prophylaxis and therapy monitoring and diagnosing active disease. Quantification of HCMV DNA may additionally have prognostic value and guide routine management. The aim of this study was to develop a reliable internally-controlled quantitative-competitive PCR (QC-PCR) for the detection and quantification of HCMV DNA viral load in peripheral blood and compare it with other methods: the HCMV pp65 antigenaemia assay in leukocyte fraction, the HCMV viraemia, both routinely employed in our laboratory, and the nucleic acid sequence-based amplification (NASBA) for detection of HCMV pp67-mRNA. METHODS: Quantitative-competitive PCR is a procedure for nucleic acid quantification based on co-amplification of competitive templates, the target DNA and a competitor functioning as internal standard. In particular, a standard curve is generated by amplifying 10(2) to 10(5) copies of target pCMV-435 plasmid with 10(4) copies of competitor pCMV-C plasmid. Clinical samples derived from 40 kidney transplant patients were tested by spiking 10(4) copies of pCMV-C into the PCR mix as internal control, and comparing results with the standard curve. RESULTS: Of the 40 patients studied, 39 (97.5%) were positive for HCMV DNA by QC-PCR. While the correlation between the number of pp65-positive cells and the number of HCMV DNA genome copies/mL and the former and the pp67mRNA-positivity were statistically significant, there was no significant correlation between HCMV DNA viral load assayed by QC-PCR and HCMV viraemia. CONCLUSIONS: The QC-PCR assay could detect from 10(2) to over 10(7) copies of HCMV DNA with a range of linearity between 10(2) and 10(5) genomes.

Adult↗
PubMed2006

Detection and monitoring of virus infections by real-time PCR.

The employment of polymerase chain reaction (PCR) techniques for virus detection and quantification offers the advantages of high sensitivity and reproducibility, combined with an extremely broad dynamic range. A number of qualitative and quantitative PCR virus assays have been described, but commercial PCR kits are available for quantitative analysis of a limited number of clinically important viruses only. In addition to permitting the assessment of viral load at a given time point, quantitative PCR tests offer the possibility of determining the dynamics of virus proliferation, monitoring of the response to treatment and, in viruses displaying persistence in defined cell types, distinction between latent and active infection. Moreover, from a technical point of view, the employment of sequential quantitative PCR assays in virus monitoring helps identifying false positive results caused by inadvertent contamination of samples with traces of viral nucleic acids or PCR products. In this review, we provide a survey of the current state-of-the-art in the application of the real-time PCR technology to virus analysis. Advantages and limitations of the RQ-PCR methodology, and quality control issues related to standardization and validation of diagnostic assays are discussed.

Computer Systems↗
PubMed2026

A photoactivatable Cre-loxP system for spatiotemporal genetic manipulation in mouse taste buds.

Conventional genetic approaches, including global gene KO and conditional KO strategies such as the Cre-loxP system, have some limitations arising from systemic effects or insufficient temporal resolution. The recently developed photoactivatable Cre (PA-Cre) system may have a potential to improve spatiotemporal control of gene manipulation. In this study, we established and validated the feasibility of the PA-Cre system using taste buds as a model. We generated TRE-PA-Cre:R26-rtTA/tdTomato mice to evaluate blue-light-induced Cre recombinase activity. Through systematic optimization of illumination parameters, we found that a single session of blue-light-illumination resulted in limited recombination efficiency, whereas a multisession illumination strategy markedly increased recombination efficiency. To further assess the utility of the PA-Cre system for gene KO, we generated TRE-PA-Cre:R26-rtTA:Tas1r3-flox mice and targeted a taste-related gene Tas1r3. Genomic DNA quantitative PCR and reverse transcription-quantitative PCR both showed partial reductions in Tas1r3 at the DNA and mRNA levels, respectively. Behavioral assays further revealed a selective decrease in sensitivity to sweet and umami stimuli. Together, these findings demonstrate PA-Cre-mediated gene manipulation in taste buds and establish a practical optical activation paradigm, providing a high-spatiotemporal-resolution tool for investigating gene function in optically targeted regions.

Animals↗
PubMed2004

Use of TaqMan 5' nuclease real-time PCR for quantitative detection of Mycoplasma genitalium DNA in males with and without urethritis who were attendees at a sexually transmitted disease clinic.

Mycoplasma genitalium is a cause of nongonococcal urethritis, particularly in patients not infected with Chlamydia trachomatis. A quantitative 5' nuclease assay (TaqMan PCR) was developed and validated. The assay detected a fragment of the MgPa adhesin gene by use of a TaqMan MGB (minor groove binder) probe and included an internal processing control to detect PCR inhibition. Urethral swab specimens and first-void urine samples from M. genitalium-positive men were examined, and the M. genitalium DNA load was correlated to symptoms and signs. The assay consistently detected <5 genome copies without cross-reactions with other mycoplasmas. Urine and urethral swab specimens from men with urethritis had higher M. genitalium DNA loads than specimens from men without urethritis. However, a very broad overlap of DNA loads between patients with and without urethritis was observed. Urethral swab specimens from patients with urethral discharge had a significantly higher DNA load than specimens from patients without discharge. This correlation was not found in first-void urine specimens.

Base Sequence↗
PubMed2006

Quantitation of immune response gene expression and cellular localisation of interleukin-1beta mRNA in Atlantic salmon, Salmo salar L., affected by amoebic gill disease (AGD).

The characterisation of selected immune response genes during amoebic gill disease (AGD) in Atlantic salmon, Salmo salar L., was performed using semi-quantitative RT-PCR, quantitative real-time RT-PCR (qRT-PCR), and in situ hybridisation (ISH). The immune response genes of interest were interleukin-1beta (IL-1beta), inducible nitric oxide synthase (iNOS), serum amyloid A (SAA), and serum amyloid P-like pentraxin (SAP). Atlantic salmon were inoculated with the ectoparasite Neoparamoeba sp., the causative agent of AGD, and gill, liver and anterior kidney tissue sampled at 0, 7 and 14 d post-inoculation (p.i.). Semi-quantitative RT-PCR was performed on the tissue samples to identify up/down-regulated mRNA expression relative to uninfected control fish and normalised to the housekeeping gene, beta-actin. Interleukin-1beta (IL-1beta) was the only immune response gene of those investigated whose mRNA was differentially regulated in any of the tissues and was found to be up-regulated in the gills by semi-quantitative RT-PCR. Increased gill IL-1beta mRNA expression was then accurately quantitated and confirmed using probe-based qRT-PCR. The cellular localisation of the IL-1beta mRNA expression in the gills of uninfected and infected fish was then determined by ISH using an IL-1beta-specific biotinylated cRNA probe. Expression of IL-1beta mRNA was localised to filament and lamellar epithelium pavement cells in gills of uninfected and infected Atlantic salmon. These data implicate the involvement of IL-1beta at the site of infection, the gills, of Atlantic salmon during AGD. This work supports previous studies that suggest IL-1beta is important in the regulation of the fish immune response to parasitic infection but additionally shows the cellular localisation of fish IL-1beta mRNA expression during infection.

Amebiasis↗
PubMed1996

Stability of hepatitis C virus RNA in serum from samples collected in a closed-tube system for serum separation and transport, as measured by a quantitative competitive PCR assay.

Recovery of hepatitis C virus (HCV) RNA, after variable time intervals from collection, was assessed using a closed-tube system for collection, separation and transport (SST tubes). Blood from four hepatitis C-infected patients was collected in 12 SST tubes and centrifuged within 1 to 3 h of collection. Tubes were then left 0, 8, 12, 24, 48 and 72 h at room temperature and at 4 degrees C before removing serum. Hepatitis C virus RNA levels were measured by quantitative polymerase chain reaction (PCR) using the AMPLICOR HVC MONITOR assay. Hepatitis C virus RNA levels in these samples were stable for at least three days at both temperatures. Polymerase chain reaction signals never decreased by more than 0.5 log. The reproducibility of the assays showed that the quantitative PCR method can be used with the storage conditions tested here. Our data suggests that processing blood in SST tubes may be very useful in following hepatitis C virus RNA titres in infected patients, especially those receiving treatment.

Hepacivirus↗
PubMed2006

Non-invasive fetal sex determination using real-time PCR.

OBJECTIVE: The aim of this study was to evaluate the specificity and sensitivity of the real-time quantitative PCR method for fetal gender determination in early pregnancy. METHODS: Blood samples were collected from 46 pregnant women prior to amniocentesis. DNA was extracted from maternal plasma using a QIAmp DNA Blood Mini Kit. DNA samples were subjected to real-time quantitative PCR amplification of SRY (as a fetus-specific marker) and beta-globin (as a marker for total plasma DNA) genes. RESULTS: The beta-globin gene sequence was detected in all samples. The SRY gene was detected in 25 of 28 plasma samples from women with male fetuses and in none of the 18 samples from women with female fetuses (sensitivity 89.2% and specificity 100%). The fetal gender was correctly determined in 43 (93.5%) of 46 maternal plasma samples. The concentration of the beta-globin gene ranged from 161 to 25,568 genome-equivalents (GE)/mL (median 1051.1), while the concentration of the SRY gene ranged from 5 to 166 GE/mL (median 27.4). The percentage of free fetal DNA ranged from 0.1% to 46.1% (median 2.0%). CONCLUSION: Amplification of fetal DNA from maternal plasma by real-time quantitative PCR is a promising method for fetal sex determination in early pregnancy. However, further studies are necessary before this procedure can be included into a clinical routine.

Chromosomes, Human, Y↗
PubMed1997

Use of quantitative competitive PCR to measure Epstein-Barr virus genome load in the peripheral blood of pediatric transplant patients with lymphoproliferative disorders.

A quantitative competitive PCR (QC-PCR) assay for Epstein-Barr virus (EBV) has been developed to provide accurate measurement of EBV genome load in pediatric transplant recipients at risk for developing posttransplant lymphoproliferative disorder (PTLD). The assay quantifies between 8 and 5,000 copies of the EBV genome in 10(5) lymphocytes after a 30-cycle amplification reaction. For 14 pediatric patients diagnosed with PTLD, the median EBV genome load was 4,000, and 13 of the 14 patients had values of >500 copies per 10(5) lymphocytes. Only 3 of 12 control transplant recipients not diagnosed with PTLD had detectable viral genome loads (median value, 40). This median was calculated by using the highest value obtained by PCR testing on each of these patients posttransplantation. PCR values of >500 copies per 10(5) lymphocytes appear to correlate with a diagnosis of PTLD. By a modified protocol, the EBV genome copy number in latently infected adults was estimated to be <0.1 copy per 10(5) lymphocytes.

Adolescent↗
PubMed2006

Comparison of three methodologies for the determination of pulmonary fungal burden in experimental murine aspergillosis.

Quantitative culture, quantitative PCR and the galactomannan enzyme immunoassay (EIA) were compared for their ability to determine the pulmonary fungal burden in a murine model of invasive aspergillosis. Quantitative culture of specimens containing hyphae under-represented the absolute fungal burden in established infection when compared with the two other methods. The best correlation was observed between the two non-culture methods. Higher variability was observed with the galactomannan EIA when compared with quantitative PCR. Collectively, these data suggest that quantitative PCR is the preferred method for determination of the pulmonary fungal burden in experimental aspergillosis.

Animals↗
PubMed2006

Comparison of a LightCycler-based real-time PCR for quantitation of Epstein-Barr viral load in different clinical specimens with semiquantitative PCR.

Measurement of viral load is important in predicting and monitoring of Epstein-Barr virus (EBV)-associated diseases especially in immunocompromised patients. The objectives of this study were the development of a LightCycler-based real-time PCR assay using primers and probes which recognize the virus capsid antigen p23-encoding region and its comparison to the semiquantitative PCR. The LightCycler protocol shows a high degree of specificity and inter- and intra-assay reproducibility. Concerning sensitivity, a good correlation between both methods was demonstrated for standard plasmid DNA, reference DNA isolated from the EBV-genome containing Namalwa cell line, and DNA extracted from plasma/cerebrospinal fluid (CSF). The detection limit was determined with 1 copy/microl eluate for the standard plasmid DNA and with 500 copies/ml plasma or CSF. For DNA derived from peripheral blood mononuclear cells (PBMCs), a decrease of sensitivity by factor 10-100 was found when larger amounts of background DNA (500 and 100 ng) were used presuming an inhibitory effect of cellular DNA. This was supported by running dilutions of the plasmid standard carried out with EBV-negative Ramos cell DNA. Thus, the cut-off level was estimated with 100-500 copies/10(5) PBMCs, when 50 or 10 ng total DNA were tested. The results indicate that the real-time PCR described here is a first line tool for the determination of viral load in plasma and CSF. Semiquantitative nested PCR is used for screening of PBMCs viral load. Positive specimens containing more than 500 copies/10(5) cells are measured for exact values by real-time PCR. To circumvent inhibitory effects of cellular DNA, measurements should be carried out generally with 50-10 ng DNA.

Capsid Proteins↗
PubMed1996

[Use of AmpliSensor to quantitate gene expression in small amounts of samples: comparison with the quantitative RT-PCR method using CCD imaging system].

Quantitative PCR methods are potentially useful for determining levels of specific gene expression and gene dosage. Previously we developed a non-radioisotopic quantitative RT-PCR method by utilizing the PCR amplification kinetics and CCD image analyzer. Recently, based on the principle of fluorescence energy transfer, the AmpliSensor system that can quantitate the PCR products concurrent to amplification in a single reaction vessel has been described. Herein, we compared the results obtained by both methods. cDNAs were synthesized from 10 human endometrial biopsy specimens. Aliquots of cDNA were used for quantitation by gel/image analyzer or AmpliSensor assay system. For estimation of the initial amount of the template(I), regression equations of the form:y = I x Ex, where y is the amount of PCR products and x is the number of cycles, were fitted to the data in the linear portion of the semi-logarithmic graphs. The relative levels of beta-actin cDNA estimated by AmpliSensor assay system was in good agreement (r = 0.91) with those of the gel/image analyzer assay system, which is cumbersome, time-consuming and needs post-amplification procedures. The AmpliSensor assay is suitable for a quantitative PCR assay based on PCR kinetics, and is applicable for diagnostic testings.

Actins↗