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Detecção de Calicivírus Humano (Small Round Structured Virus-SRSV) pela Relação em Cadeia da Polimerase( PCR) em Ostras do Litoral do Estado de São Paulo.; Detection of human calicivirus (Small round structured virus - SRSV) using polymerase chain reaction (PCR) in oysters from São Paulo beaches, Brazil.

Vieira, Luiz Carlos
Fonte: Biblioteca Digitais de Teses e Dissertações da USP Publicador: Biblioteca Digitais de Teses e Dissertações da USP
Tipo: Dissertação de Mestrado Formato: application/pdf
Publicado em 28/06/1999 Português
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Vírus causadores de gastroenterite, descritos como pequenos vírus de estrutura arredondada (Small Round Structured Viruses-SRSV), foram detectados em extratos de ostras Crassostrea spp., coletadas em regiões distintas do litoral do Estado de São Paulo, utilizando a Reação em Cadeia por Polimerase com transcrição reversa (RT-PCR).Treze lotes de amostras, contendo 10 g de estômagos e divertículos, foram processados para extração e concentração das partículas virais, mediante adsorção-eluição e precipitação por polietilenoglicol (PEG 6000), e purificação com fluorocarbono (Freon, Dupont Co.) para eliminação prévia dos inibidores da reação de RT-PCR. A extração do ácido nucleico viral, foi realizada com uma mistura de isotiocianato de guanidina e fenol (TRIzol ®, Gibco) e clorofórmio, sendo completada com uma purificação em coluna spin, com membrana de polissulfona (Millipore,Corp.). A reação de RT-PCR foi realizada em uma única etapa, utilizando o kit 'Super Script ONE-STEP RT-PCR System'® (Gibco). A reação de amplificação enzimática revelou, por eletroforese em gel de agarose (2%), corada com brometo de etídio, produtos de 123 bp, em 3 (23%) dos 13 lotes de amostras coletadas durante o verão de 1998...

Detecção do vírus respiratório sincicial humano (HRSV) pela RT-PCR em tubo único, em amostras clínicas ; Single-Tube Reverse Transcriptase Polymerase Chain Reaction for diagnosis of Human Respiratory Syncytial Virus (HRSV) in clinical samples

Nascimento, Cesar Augusto do
Fonte: Biblioteca Digitais de Teses e Dissertações da USP Publicador: Biblioteca Digitais de Teses e Dissertações da USP
Tipo: Dissertação de Mestrado Formato: application/pdf
Publicado em 09/06/2006 Português
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126.02%
O vírus respiratório sincicial humano (HRSV) é principal agente causador de infecções do trato respiratório inferior em crianças e lactentes. Um diagnóstico rápido e preciso evitaria o uso desnecessário de antibióticos, nos casos em que a infecção é viral. A reação em cadeia da polimerase após transcrição reversa (RT-PCR) e o ensaio de imunofluorescência indireta (IFI) são considerados ferramentas importantes na detecção do HRSV, pela alta sensibilidade e especificidade. Visando simplificar e minimizar os riscos de contaminação freqüentes, em duas etapas, foi padronizada uma reação em tubo único para detecção do HRSV em amostras clínicas. Aspirados de nasofaringe de 226 crianças de 0-5 anos de idade, com doença respiratória, atendidas no Hospital Universitário da Universidade de São Paulo (HU-USP), foram testados por imunofluorescência indireta, RT semi Nested PCR e RT-PCR em tubo único. Cento e duas amostras (45,1%) foram positivas em pelo menos uma das técnicas e 75 (33,2%) em todas. Três (1,3%) amostras foram positivas por IFI e RT semi Nested PCR, 1 (0,4%) foi positiva por IFI e RT-PCR em tubo único, 5 (2,2%) amostras foram positivas somente por IFI, 2 (0,9%) somente por RT semi Nested PCR e 16 (7...

Use of reverse transcriptase polymerase chain reaction (RT-PCR) in molecular screening of Newcastle disease virus in poultry and free-living bird populations

de Oliveira Torres Carrasco, Adriano; Rodrigues, Juliana Nogueira Martins; Seki, Meire Christina; de Moraes, Fabricio Edgar; Silva, Jaqueline Raymondi; Durigon, Edison Luis; Pinto, Aramis Augusto
Fonte: Universidade Estadual Paulista Publicador: Universidade Estadual Paulista
Tipo: Artigo de Revista Científica Formato: 569-576
Português
Relevância na Pesquisa
156.22%
The aim of this study was to evaluate a simple molecular method of reverse transcriptase polymerase chain reaction (RT-PCR) to differentiate Newcastle disease virus strains according to their pathogenicity, in order to use it in molecular screening of Newcastle disease virus in poultry and free-living bird populations. Specific primers were developed to differentiate LaSota-LS-(vaccine strain) and Sao Joao do Meriti-SJM-strain (highly pathogenic strain). Chickens and pigeons were experimentally vaccinated/infected for an in vivo study to determine virus shedding in feces. Validation of sensitivity and specificity of the primers (SJM and LS) by experimental models used in the present study and results obtained in the molecular analysis of the primers by BLAST made it possible to generalize results. The development of primers that differentiate the level of pathogenicity of NDV stains is very important, mainly in countries where real-time RT-PCR is still not used as a routine test. These primers were able to determine the presence of the agent and to differentiate it according to its pathogenicity. © 2012 Springer Science+Business Media B.V.

Analysis of Respiratory Syncytial Virus in Clinical Samples by Reverse Transcriptase-Polymerase Chain Reaction Restriction Mapping

Fonte: Instituto Oswaldo Cruz, Ministério da Saúde Publicador: Instituto Oswaldo Cruz, Ministério da Saúde
Tipo: Artigo de Revista Científica Formato: text/html
Publicado em 01/05/1997 Português
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115.96%
The aim of this study was to develop a polymerase chain reaction (PCR) for the detection of respiratory syncytial virus (RSV) genomes. The primers were designed from published sequences and selected from conserved regions of the genome encoding for the N protein of subgroups A and B of RSV. PCR was applied to 20 specimens from children admitted to the respiratory ward of "William Soler" Pediatric Hospital in Havana City with a clinical diagnosis of bronchiolitis. The PCR was compared with viral isolation and with an indirect immunofluorescence technique that employs monoclonal antibodies of subgroups A and B. Of 20 nasopharyngeal exudates, 10 were found positive by the three assayed methods. In only two cases, samples that yielded positive RNA-PCR were found negative by indirect immunofluorescence and cell culture. Considering viral isolation as the "gold standard" technique, RNA-PCR had 100% sensitivity and 80% specificity. RNA-PCR is a specific and sensitive technique for the detection of the RSV genome. Technical advantages are discussed

A Simple Reverse Transcription-Polymerase Chain Reaction for Dengue Type 2 Virus Identification

Fonte: Instituto Oswaldo Cruz, Ministério da Saúde Publicador: Instituto Oswaldo Cruz, Ministério da Saúde
Tipo: Artigo de Revista Científica Formato: text/html
Publicado em 01/05/1997 Português
Relevância na Pesquisa
126.11%
We show here a simplified reverse transcription-polymerase chain reaction (RT-PCR) for identification of dengue type 2 virus. Three dengue type 2 virus strains, isolated from Brazilian patients, and yellow fever vaccine 17DD, as a negative control, were used in this study. C6/36 cells were infected with the virus, and tissue culture fluids were collected after 7 days of infection period. The RT-PCR, a combination of RT and PCR done after a single addition of reagents in a single reaction vessel was carried out following a digestion of virus with 1% Nonidet P-40. The 50ml assay reaction mixture included 50 pmol of a dengue type 2 specific primer pair amplifying a 210 base pair sequence of the envelope protein gene, 0.1 mM of the four deoxynucleoside triphosphates, 7.5U of reverse transcriptase, and 1U of thermostable Taq DNA polymerase. The reagent mixture was incubated for 15 min at 37oC for RT followed by a variable amount of cycles of two-step PCR amplification (92oC for 60 sec, 53oC for 60 sec) with slow temperature increment. The PCR products were subjected to 1.7% agarose gel electrophoresis and visualized with UV light after gel incubation in ethidium bromide solution. DNA bands were observed after 25 and 30 cycles of PCR. Virus amount as low as 102.8 TCID50/ml was detected by RT-PCR. Specific DNA amplification was observed with the three dengue type 2 strains. This assay has advantages compared to other RT-PCRs: it avoids laborious extraction of virus RNA; the combination of RT and PCR reduces assay time...

Diagnosis of Dengue by Using Reverse Transcriptase-Polymerase Chain Reaction

Fonte: Instituto Oswaldo Cruz, Ministério da Saúde Publicador: Instituto Oswaldo Cruz, Ministério da Saúde
Tipo: Artigo de Revista Científica Formato: text/html
Publicado em 01/09/1997 Português
Relevância na Pesquisa
136.09%
A rapid identification of dengue viruses from clinical samples by using a nested reverse transcriptase-polymerase chain reaction (RT-PCR) procedure was carried out for diagnostic and epidemiological purposes. RT-PCR identified DEN-1 and DEN-2 viruses in 41% (41/100) of previously confirmed cases and provided an accurate confirmation of DHF in four fatal cases. RT-PCR was also useful for detecting and typing dengue viruses in suspected cases, allowing a rapid identification of new serotypes in endemic areas

Relevance of micrometastases detected by reverse transcriptase-polymerase chain reaction for melanoma recurrence: systematic review and meta-analysis

Silva,Allisson Monteiro da; Oliveira Filho,Renato Santos de; Ferreira,Lydia Masako; Saconato,Humberto
Fonte: Associação Paulista de Medicina - APM Publicador: Associação Paulista de Medicina - APM
Tipo: Artigo de Revista Científica Formato: text/html
Publicado em 01/01/2003 Português
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156.48%
CONTEXT: Cutaneous melanoma presents significant morbidity and mortality. Nowadays, about 90% of them are diagnosed by clinical examination and most are localized melanomas. Sentinel node biopsy has brought about a new and interesting approach towards localized cutaneous melanoma. The meaning of micrometastases in sentinel nodes diagnosed by the reverse transcriptase-polymerase chain reaction is not well established. OBJECTIVE: To define the real value of micrometastases diagnosed by the reverse transcriptase-polymerase chain reaction in relation to melanoma recurrence. METHODS: Systematic literature review and meta-analysis. The Cochrane Library, Medline, Embase and Lilacs were the databases searched. We used the following key words: sentinel node and melanoma; sentinel node and reverse transcriptase-polymerase chain reaction; melanoma and reverse transcriptase-polymerase chain reaction. Cohort studies enrolling localized cutaneous melanoma patients who underwent sentinel node biopsy were selected. Sentinel node evaluations included hematoxylin and eosin, immunohistochemistry and reverse transcriptase-polymerase chain reaction. RESULTS: Out of the 1,542 studies evaluated, four were eligible. The four studies, when combined, were statistically homogeneous. The sample totaled 450 patients grouped as follows: 163 with a sentinel node negative to hematoxylin eosin and immunohistochemistry and positive to the reverse transcriptase-polymerase chain reaction; 192 with a sentinel node negative to hematoxylin eosin...

Vitiligo: analysis of grafting versus curettage alone, using melanocyte morphology and reverse transcriptase polymerase chain reaction for tyrosinase mRNA

Machado Filho,Carlos D’Aparecida dos Santos; Almeida,Fernando Augusto; Proto,Rodrigo Sestito; Landman,Gilles
Fonte: Associação Paulista de Medicina - APM Publicador: Associação Paulista de Medicina - APM
Tipo: Artigo de Revista Científica Formato: text/html
Publicado em 01/01/2005 Português
Relevância na Pesquisa
136.09%
CONTEXT AND OBJECTIVE: Recent studies have indicated that vitiligo areas contain inactive or dormant melanocytes. Melanin synthesis is related to tyrosinase presence and indicative of active metabolic state. The aim of this study was to compare repigmentation, epidermal melanocyte distribution and tyrosinase mRNA detection through reverse transcriptase polymerase chain reaction, in tissue samples of vitiligo, before and after curettage, with or without subsequent autologous skin graft using a new method. DESIGN AND SETTING: Prospective, in the Department of Dermatology, Faculdade de Medicina do ABC, Santo André. METHODS: Two vitiligo areas were curetted. One subsequently received grafted normal sacral autologous skin, whereas the other had no further treatment. The curetted areas were examined after 30 days, to evaluate the degree of repigmentation. The melanocyte percentages and tyrosinase mRNA presence in normal skin and vitiligo areas, before and after curettage and grafting, were compared. RESULTS: Complete repigmentation was seen in all grafted areas, whereas non-grafted curetted vitiligo presented partial repigmentation. The melanocyte percentage in grafted areas was greater than in non-treated vitiligo skin (p = 0.01) and skin with curettage alone (p = 0.015). Tyrosinase mRNA was negative in 93.75% of non-treated vitiligo areas. After treatment (curettage alone or curettage and grafting)...

A comparative analysis of the suitability of different peripheral blood samples for reverse transcriptase polymerase chain reaction

Melo,Adriene Siqueira de; Lorena,Virginia Maria Barros de; Braz,Suellen Carvalho de Moura; Gomes,Yara de Miranda
Fonte: Associação Brasileira de Hematologia e Hemoterapia e Terapia Celular Publicador: Associação Brasileira de Hematologia e Hemoterapia e Terapia Celular
Tipo: Artigo de Revista Científica Formato: text/html
Publicado em 01/01/2010 Português
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156.16%
Venipuncture is one of the easiest clinical procedures to obtain viable blood samples to evaluate gene expression using mRNA analysis. However, the use of this sample type in reverse transcriptase polymerase chain reaction tests (RT-PCR) without prior treatment is controversial. We therefore propose to compare the suitability of different peripheral blood samples (whole blood without treatment, whole blood with hemolysis, peripheral blood mononuclear cells and frozen whole blood) for RT-PCR analysis. The results showed that, despite the blood sample being peripheral, it is possible to extract a fair amount of RNA and perform target gene amplification. Thus, peripheral blood without prior treatment could be used to investigate the gene expression using Real Time PCR.

Rapid detection and typing of dengue viruses from clinical samples by using reverse transcriptase-polymerase chain reaction.

Lanciotti, R S; Calisher, C H; Gubler, D J; Chang, G J; Vorndam, A V
Fonte: PubMed Publicador: PubMed
Tipo: Artigo de Revista Científica
Publicado em /03/1992 Português
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106.1%
We report on the development and application of a rapid assay for detecting and typing dengue viruses. Oligonucleotide consensus primers were designed to anneal to any of the four dengue virus types and amplify a 511-bp product in a reverse transcriptase-polymerase chain reaction (PCR). First, we produced a cDNA copy of a portion of the viral genome in a reverse transcriptase reaction in the presence of primer D2 and then carried out a standard PCR (35 cycles of heat denaturation, annealing, and primer extension) with the addition of primer D1. The resulting double-stranded DNA product of the RT-PCR was typed by two methods: dot blot hybridization of the 511-bp amplified product to dengue virus type-specific probes or a second round of PCR amplification (nested PCR) with type-specific primers, yielding DNA products the unique sizes of which were diagnostic for each dengue virus serotype. The accumulated data demonstrated that dengue viruses can be accurately detected and typed from viremic human serum samples.

Expression of Angiotensin AT1 and AT2 Receptors in Adult Rat Cardiomyocytes after Myocardial Infarction : A Single-Cell Reverse Transcriptase-Polymerase Chain Reaction Study

Busche, Silke; Gallinat, Stefan; Bohle, Rainer-Maria; Reinecke, Alexander; Seebeck, Jörg; Franke, Folker; Fink, Ludger; Zhu, Mingyan; Sumners, Colin; Unger, Thomas
Fonte: American Society for Investigative Pathology Publicador: American Society for Investigative Pathology
Tipo: Artigo de Revista Científica
Publicado em /08/2000 Português
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106.14%
The effector hormone of the renin-angiotensin system, angiotensin II, plays a major role in cardiovascular regulation. In rats, both angiotensin receptor subtypes, AT1 and AT2, are up-regulated after myocardial infarction but previous studies failed to identify the cell types which express the AT2 receptor in the heart. To address this question we established a single-cell reverse transcriptase-polymerase chain reaction for AT1 and AT2 receptors to determine whether these receptor subtypes are expressed in adult rat cardiomyocytes before and 1 day after myocardial infarction. By laser-assisted cell picking, section profiles of single cells without genomic DNA contamination were isolated. After dividing samples into two identical aliquots, polymerase chain reaction amplification for AT1 and AT2 receptors was carried out and polymerase chain reaction products were subjected to gel electrophoresis. Compared to control (n = 4) and sham-operated animals (n = 4), the number of cardiomyocytes expressing the AT1 receptor mRNA 1 day after myocardial infarction (n = 4) was not changed (42% and 33% versus 45%, respectively). On the other hand, AT2 receptor mRNA was expressed in 8% and 13%, respectively, of cardiomyocytes gained from control (n = 4) and sham-operated animals (n = 4) and in 14% isolated after myocardial infarction (n = 4). These results demonstrate for the first time that the AT2 receptor is expressed in adult cardiomyocytes in vivo. They further suggest that the previously observed up-regulation of cardiac AT1 and AT2 receptors after myocardial infarction involves cell types other than cardiomyocytes.

Amplification of the t(2; 13) and t(1; 13) translocations of alveolar rhabdomyosarcoma in small formalin-fixed biopsies using a modified reverse transcriptase polymerase chain reaction.

Anderson, J.; Renshaw, J.; McManus, A.; Carter, R.; Mitchell, C.; Adams, S.; Pritchard-Jones, K.
Fonte: PubMed Publicador: PubMed
Tipo: Artigo de Revista Científica
Publicado em /02/1997 Português
Relevância na Pesquisa
106.16%
Detection of characteristic chromosomal translocations has aided diagnosis of the small round cell tumors of childhood and may help to stratify patients into clinical groups. The detection of the abnormalities by classical cytogenetic techniques has been supplemented by fluorescent in situ hybridization and reverse transcriptase polymerase chain reaction (RT-PCR). These techniques allow diagnoses to be made using only very small amounts of tumor tissue. We here describe a technique for the rapid and specific detection by modified reverse transcriptase polymerase chain reaction of characteristic chromosomal translocations of alveolar rhabdomyosarcoma with small amounts of formalin-fixed tissue as the starting material. Of 27 samples studied, 4 cases are described in which the detection of translocations by this method cast doubt on the original histopathological diagnosis. These cases demonstrate the critical diagnostic importance of the detection of these translocations in rhabdomyosarcoma.

JAZF1/JJAZ1 Gene Fusion in Endometrial Stromal Sarcomas: Molecular Analysis by Reverse Transcriptase-Polymerase Chain Reaction Optimized for Paraffin-Embedded Tissue

Hrzenjak, Andelko; Moinfar, Farid; Tavassoli, Fattaneh A.; Strohmeier, Bettina-; Kremser, Marie-Luise; Zatloukal, Kurt; Denk, Helmut
Fonte: American Society for Investigative Pathology Publicador: American Society for Investigative Pathology
Tipo: Artigo de Revista Científica
Publicado em /08/2005 Português
Relevância na Pesquisa
106.16%
Endometrial stromal tumors are rare uterine neoplasms including benign stromal nodules, low-grade endometrial stromal sarcomas (ESS), and undifferentiated endometrial sarcomas (UES), the latter representing the most aggressive form. Morphological characteristics and cytogenetic abnormalities are heterogeneous, making diagnosis difficult. Recently, a gene fusion on chromosome 7 that includes two zinc-finger genes (JAZF1 and JJAZ1) has been discovered in these tumors. Hitherto only 31 cases, described by three different research groups, have shown JAZF1/JJAZ1 fusion in ∼50% of all analyzed low-grade ESSs whereas it is less frequent in UESs. In this study we analyzed 20 ESS and 2 UES cases using two-step reverse transcriptase-polymerase chain reaction optimized for formalin-fixed, paraffin-embedded tissue. In our subset of samples, the JAZF1/JJAZ1 fusion transcript occurred in 80% of analyzed ESS cases and in none of two UES cases. In comparison to published data, our results identified the JAZF1/JJAZ1 gene fusion more frequently in endometrial stromal tumors than hitherto presumed. This cytogenetic abnormality was not present in normal endometria, leiomyomas, or leiomyosarcomas or in lung, gastric, or hepatic carcinomas, indicating its specificity for endometrial stromal tumors. In combination with other established methods...

Critical Evaluation of Real-Time Reverse Transcriptase-Polymerase Chain Reaction for the Quantitative Detection of Cytokeratin 20 mRNA in Colorectal Cancer Patients

Dandachi, Nadia; Balic, Marija; Stanzer, Stefanie; Halm, Michael; Resel, Margit; Hinterleitner, Thomas Anton; Samonigg, Hellmut; Bauernhofer, Thomas
Fonte: American Society for Investigative Pathology Publicador: American Society for Investigative Pathology
Tipo: Artigo de Revista Científica
Publicado em /11/2005 Português
Relevância na Pesquisa
106.17%
We evaluated the usefulness of cytokeratin 20 (CK20) mRNA expression in the quantitative detection of circulating tumor cells in the blood of patients with colorectal cancer (CRC). Blood samples from healthy volunteers (HVs; n = 37), patients with localized (n = 42) and metastatic colorectal cancer (n = 40), and patients with chronic inflammatory bowel disease (CID; n = 15) were examined. After immunomagnetic enrichment using microbeads against human epithelial antigen, total RNA was extracted, reverse transcribed, and analyzed by real-time reverse transcriptase-polymerase chain reaction using the LightCycler instrument. CK20 expression in peripheral blood was found in 46 of 82 (56%) patients with CRC, 8 of 37 (22%) HVs, and 9 of 15 (60%) patients with CID. Levels of CK20 mRNA were significantly higher in blood samples from CRC patients (median 681) than in blood samples from HVs (median 0) (P = 0.001), whereas no difference could be detected between patients with CRC and CID. Although the present technique could not distinguish CRC from CID, the method warrants further efforts to improve sample preparation and tumor cell enrichment, which may render real-time CK20 reverse transcriptase-polymerase chain reaction a feasible technique in identifying circulating tumor cells in peripheral blood of cancer patients.

Towards Quantitative mRNA Analysis in Paraffin-Embedded Tissues Using Real-Time Reverse Transcriptase-Polymerase Chain Reaction : A Methodological Study on Lymph Nodes from Melanoma Patients

Abrahamsen, Helene Nortvig; Steiniche, Torben; Nexo, Ebba; Hamilton-Dutoit, Stephen J.; Sorensen, Boe Sandahl
Fonte: American Society for Investigative Pathology Publicador: American Society for Investigative Pathology
Tipo: Artigo de Revista Científica
Publicado em /02/2003 Português
Relevância na Pesquisa
106.2%
Improved extraction techniques combined with sensitive real-time reverse transcriptase-polymerase chain reaction may allow detection of mRNA in formalin-fixed, paraffin-embedded (FFPE) materials, but the factors affecting mRNA quantification in clinical material using these methods have not been systematically analyzed. We designed analyses using real-time reverse transcriptase-polymerase chain reaction for quantification of MART-1, β-actin, and β2-microglobulin mRNAs. The analytical intra- and interassay imprecision (coefficient of variation) was in the range 10 to 20% for all three genes studied. Using these protocols, we studied the influence of tissue autolysis and length of formalin-fixation on mRNA detection in metastatic melanoma. Delay in freezing reduced detectable mRNA, although this was less than predicted and mostly occurred early in autolysis. MART-1, β-actin, and β2-microglobulin mRNAs were consistently detected in FFPE metastatic melanoma even after fixation for up to 3 weeks, although the total mRNA detected was markedly reduced in fixed compared with fresh tissues (up to 99%). Quantification of MART-1 was, however, possible if this was expressed relative to a housekeeping gene. The polymerase chain reaction product from FFPE tissues could be increased up to 100-fold amplifying short (<136 bp) compared with long amplicons. Variations in time before tissue processing and in fixation length seem to be less important sources of imprecision than previously assumed. Our findings suggest that quantitative analysis of mRNA in archive and routine diagnostic tissues may be possible.

Detection of occult metastasis in lymph nodes from colorectal cancer patients: a multiple-marker reverse transcriptase-polymerase chain reaction study

Chen, G.; McIver, C.; Texler, M.; Lloyd, J.; Rieger, N.; Hewett, P.; sen Wan, D.; Hardingham, J.
Fonte: Lippincott Williams & Wilkins Publicador: Lippincott Williams & Wilkins
Tipo: Artigo de Revista Científica
Publicado em //2004 Português
Relevância na Pesquisa
156.38%
INTRODUCTION Lymph node status is a key factor for disease staging and is the main determinant for adjuvant therapy of colorectal cancer. The current staging procedure is unable to identify occult metastasis in lymph nodes, which is likely to be an important cause of treatment failure in some early-stage patients. The detection of occult metastasis could identify a patient subgroup at risk for disease relapse that would benefit from adjuvant therapy. The purpose of this study was to establish and test a multimarker reverse transcriptase-polymerase chain reaction assay for the molecular detection of occult metastases in lymph nodes. METHODS Forty-four patients with colorectal cancer and 14 patients with benign bowel diseases undergoing colonic resection were enrolled in the study. Reverse transcriptase-polymerase chain reaction was used to detect expression of three epithelial markers, carcinoembryonic antigen, cytokeratin 20, and guanylyl cyclase C, in fresh colorectal lymph node tissue. RESULTS Forty-six of 47 (97.9 percent) histologically positive lymph nodes were also positive by reverse transcriptase-polymerase chain reaction. Of 221 histologically negative nodes, 97 (43.9 percent) were positive for at least one of the three markers by reverse transcriptase-polymerase chain reaction: 24.9 percent for carcinoembryonic antigen...

Quantification of mRNA for the vitamin D metabolizing enzymes CYP27B1 and CYP24 and vitamin D receptor in kidney using real-time reverse transcriptase-polymerase chain reaction

Anderson, P.; O'Loughlin, P.; May, B.; Morris, H.
Fonte: Soc Endocrinology Publicador: Soc Endocrinology
Tipo: Artigo de Revista Científica
Publicado em //2003 Português
Relevância na Pesquisa
156.18%
Critical to an understanding of the control of 1,25-dihydroxyvitamin D (1,25D) activity is a molecular appreciation of the regulation of three genes, 25-hydroxyvitamin D-1alpha-hydroxylase (CYP27B1), 25-hydroxyvitamin D-24-hydroxylase (CYP24) and vitamin D receptor (VDR). We now report the sensitivity, reproducibility and accuracy of a real-time reverse transcriptase-polymerase chain reaction protocol (Taqman) for the quantification of mRNA levels for these genes in total RNA extracted from kidney tIssue. The sensitivity of the protocol was at least 150 copies of mRNA per reaction. Reproducibility, expressed as the coefficient of variation, ranged between 14 and 30% at the level of approximately 10(4) copies of mRNA per reaction. Accuracy was estimated at greater than 95% for each of these mRNAs. This protocol allows for the comparison of absolute mRNA levels in extracted total RNA in kidneys from animals fed diets containing different levels of calcium, ranging from 0.05% to 1%. Serum 1,25D levels were decreased when the dietary calcium concentration was increased (P<0.05). The levels of CYP27B1 mRNA were highest in the animals fed the 0.05% calcium diet (P<0.01). Conversely, CYP24 and VDR mRNA levels were highest in the animals fed the 1% calcium diet (P<0.01). Both CYP27B1 and CYP24 mRNA levels were major determinants of serum 1...

Clinical significance of human telomerase reverse transcriptase in patients with colon cancer

Li, L.; Wan, D.S.; Pan, Z.; Hardingham, J.; Rieger, N.; Hewett, P.; Zhou, Y.; Chen, G.
Fonte: Zhongshan Yike Daxue, Zhongliuzhi Zhongxin Publicador: Zhongshan Yike Daxue, Zhongliuzhi Zhongxin
Tipo: Artigo de Revista Científica
Publicado em //2004 Português
Relevância na Pesquisa
126.21%
BACKGROUND & OBJECTIVE: Although telomerase activity can be detected in 70%-90% malignant tumor tissues, it is still a controversial prognostic factor of patients with malignant tumors. This study was to evaluate clinical significance of human telomerase reverse transcriptase (hTERT) in patients with colon cancer. METHODS: Expression of hTERT in 59 matched pairs of tumor and adjacent non-tumorous mucosa samples from patients with colon cancer who underwent complete resection were measured by quantitative real-time reverse transcriptase-polymerase chain reaction (RT-PCR). RESULTS: Expression of hTERT in both tumor samples and nontumorous mucosa samples has no significant correlation with clinicopathologic factors. Of 32 patients with colon cancer of Dukes' A, and B stages, prognosis of 18 (56%) patients with hTERT expression of lower than 0.60 in tumor tissue was better than that of 27 (44%) patients with hTERT expression of higher than 0.60 (P=0.006), prognosis of 20 (62%) patients with the value of hTERT expression in tumor tissue subtracted from that in matched non-tumorous tissue below 0.50 was better than that of 12 (38%) patients with that value above 0.50 (P=0.035). In 27 patients with colon cancer of Dukes' C, and D stages, it was not practical to estimate the patients' prognosis with hTERT expression level in tumor tissue and the expression difference between tumor tissue and non-tumorous tissue. CONCLUSION: hTERT expression may be a potential prognostic index for patients with colon cancer of Dukes' A...

Detection of chimeric transcripts in desmoplastic small round cell tumor and related developmental tumors by reverse transcriptase polymerase chain reaction. A specific diagnostic assay.

de Alava, E.; Ladanyi, M.; Rosai, J.; Gerald, W. L.
Fonte: PubMed Publicador: PubMed
Tipo: Artigo de Revista Científica
Publicado em /12/1995 Português
Relevância na Pesquisa
106.16%
Desmoplastic small round cell tumor is a recently described entity associated with fusion of the EWS and WT1 genes and with expression of a chimeric transcript. To investigate the structure and potential diagnostic utility of the detection of EWS-WT1 chimeric RNA in desmoplastic small round cell tumor, 12 examples of this entity and 49 other tumors that enter in its differential diagnosis were studied by reverse transcriptase polymerase chain reaction for the presence of EWS-WT1, EWS-FLI-1, PAX3-FKHR, and PAX7-FKHR chimeric transcripts. EWS-WT1 was detected in 11 of 12 desmoplastic small round cell tumors but not in any other tumor type studied, including 17 Wilms' tumors, 10 Ewing's sarcomas/primitive neuroectodermal tumors, 13 alveolar rhabdomyosarcomas, and 9 embryonal rhabdomyosarcomas. One desmoplastic small round cell tumor was found to have a variant EWS-WT1 chimeric product that included exon 8 of EWS EWS-FLI-1 chimeric RNA was present in all Ewing's sarcoma/primitive neuroectodermal tumor and not identified in any other tumor types, including desmoplastic small round cell tumor. PAX3/PAX7-FKHR chimeras were present in 9 of 13 alveolar rhabdomyosarcomas but not in any other tumors. Detection of chimeric transcripts by reverse transcriptase polymerase chain reaction is a very specific aid in differential diagnosis of developmental tumors and further establishes desmoplastic small round cell tumor as a distinct entity.

Real-time reverse transcription polymerase chain reaction; Reação em cadeia da polimerase da transcrição reversa em tempo real

Ladeira, Pedro Ribeiro Soares de; Isaac, Cesar; Ferreira, Marcus Castro
Fonte: Universidade de São Paulo. Faculdade de Medicina Publicador: Universidade de São Paulo. Faculdade de Medicina
Tipo: info:eu-repo/semantics/article; info:eu-repo/semantics/publishedVersion; Formato: application/pdf; application/pdf
Publicado em 01/03/2011 Português
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RT-qPCR is one of the most widespread and reliable methods of measuring gene expression. First, the extraction of mRNA from a homogeneous cell group is done in order to perform the RT phase (reverse transcription). At this point the reverse transcriptase enzyme synthesizes cDNA corresponding to each RNA strand. Then begins the qPCR (real time polymerase chain reaction), which gets its name because it gives quantitative results (“q” of qPCR), unlike the conventional PCR qualitative results. This stage is characterized by amplification of a specific site in the set of cDNA, which is achieved, briefly, through the use of a DNA-dependent thermostable DNA polymerase (Taq polymerase), two oligomers specific to serve as primers for polymerase and a non-specific DNA fluorophore (SYBR Green I, for example). As the double-strand cDNA is being synthesized, the fluorophore binds to its strands and, after being excited, emits light in proportion to the number of double chains. Through graphical analysis, it is possible to quantify the initial sample of cDNA that, in absence of errors, is proportional to mRNA; RT-qPCR é um dos métodos de mensurar expressão gênica mais difundidos e confiáveis utilizados atualmente. Primeiro, realiza-se extração do RNAm de um grupo celular homogêneo para poder realizar a fase RT (transcrição reversa). Neste momento a enzima transcriptase reversa sintetiza o DNAc correspondente de cada fita de RNA. Em seguida inicia-se a qPCR (reação em cadeia da polimerase em tempo real)...