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Results for “Reverse Transcriptase Polymerase Chain Reaction”

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At least 217 records · Page 12Linked to original sources
PubMed1998

Detection of chimaeric transcripts of the immunoglobulin heavy chain and BCL6 genes by reverse-transcriptase polymerase chain reaction in B-cell non-Hodgkin's lymphomas.

T(3;14)(q27;q32) is frequently detected in B-cell non-Hodgkin's lymphomas, especially the diffuse large cell type and the follicular type. The BCL6 gene encoding a putative transcriptional factor which resides on 3q27 rearranges to the immunoglobulin heavy chain (IgH) gene on 14q32 in this chromosomal translocation. The upstream regulatory region of the BCL6 gene is replaced by the IgH gene. Deregulation of the BCL6 gene may contribute to tumourigenesis of these diseases. The rearrangement between the IgH and BCL6 genes generates chimaeric transcripts in which the joining (J) region of the IgH gene fuses to exon 3 of the BCL6 gene. We established a method to detect these chimaeric transcripts by reverse transcriptase polymerase chain reaction (RT-PCR) using the consensus sequence of the J region and the sequence of exon 3 of the BCL6 gene as primers. Using the semi-nested RT-PCR method and a cell line carrying t(3;14)(q27;q32), we detected one lymphoma cell among 10,000 background cells. We detected these chimaeric transcripts in two out of 13 clinical samples by this method. This method can detect t(3;14)(q27;q32) easily, whereas this alteration is frequently overlooked by routine karyotype analysis. Since this technique is sensitive enough to detect a small number of lymphoma cells with this genetic abnormality, it could be employed to detect contaminating lymphoma cells in bone marrow and peripheral blood and minimal residual diseases.

Base Sequence↗
PubMed1993

Biological variation and quality control of plasma human immunodeficiency virus type 1 RNA quantitation by reverse transcriptase polymerase chain reaction.

Quantitation of human immunodeficiency virus type 1 (HIV-1) RNA in the plasma of seropositive individuals was performed by using an external control assay with techniques to standardize and control each measurement. Rigorous study of the variability of the assay showed that the median intraassay reproducibility was log10 0.15 RNA copies per ml of plasma, while the median interassay reproducibility on replicate plasma samples was log10 0.25 copies perml. Specimen stability studies showed reproducible recovery of RNA from plasma stored at -70 degrees C for up to 12 months. In clinically stable patients who were either untreated or taking zidovudine, the average week-to-week variation in plasma RNA levels, measured in real time, was log10 0.30 RNA copies per ml. In contrast, patients either initiating or changing antiretroviral therapy showed a fall of log10 0.8 to log10 2.0 copies per ml in plasma RNA levels. Overall, 105 of 110 (96%) HIV-1-seropositive individuals with CD4 counts of 36 to 868 cells per mm3 had quantifiable HIV-1 RNA over a range of log10 2.70 to log10 6.23 RNA copies per ml, including 81% (13 of 16) of the individuals with greater than 500 CD4 cells per mm3. Accurate and reproducible quantitation of plasma viremia in real time by reverse transcriptase polymerase chain reaction, particularly in asymptomatic HIV-1-infected individuals with high CD4 counts, provides a basis for the use of this virologic measure to monitor the short- and long-term effects of early intervention therapeutic strategies on viral burden.

CD4-Positive T-Lymphocytes↗
PubMed1996

Detection of t(2;5)(p23;q35) translocation by reverse transcriptase polymerase chain reaction and in situ hybridization in CD30-positive primary cutaneous lymphoma and lymphomatoid papulosis.

The t(2;5) generates a chimeric NPM-ALK transcript encoded by the nucleophosmin NPM gene fused to the anaplastic lymphoma kinase gene ALK. Using a reverse transcriptase nested polymerase chain reaction assay we have detected NPM-ALK transcripts within CD30+ primary cutaneous lymphoma and lymphomatoid papulosis (LP). The t(2;5) was identified in 4 out of 9 CD30+ anaplastic lymphomas and in 1 out of 4 CD30+ pleomorphic lymphomas. Moreover, the t(2;5) was detected in 3 out of 10 LPs. All NPM-ALK-positive lymphomas and 1 NPM-ALK-positive LP exhibited a clonal rearrangement of the T cell receptor gamma-chain gene. The t(2;5) was detected in 2 cases of LP without other evidence for a clonal lymphoid population. To identify cells carrying the t(2;5) translocation, we used immunohistochemistry to detect the ALK-encoded p80 protein and in situ hybridization for the specific detection of NPM-ALK transcripts. Both p80 protein and NPM-ALK transcripts were expressed by anaplastic or large CD30+ lymphoma cells with positive NPM-ALK amplification. The presence of t(2;5) in a subset of CD30+ cutaneous lymphoma and LP may indicate a common pathogenesis with a subset of anaplastic nodal lymphoma.

Adult↗
PubMed1992

Studies on basic fibroblast growth factor (FGF-beta) gene expression in the rat and pig ovary using in situ hybridization and quantitative reverse transcriptase--polymerase chain reaction techniques.

In order to gain further understanding of the physiology of basic fibroblast growth factor (FGF-beta) in the mammalian reproductive tract, the expression of FGF-beta mRNA in the rat and porcine ovary has been examined by in situ hybridization and quantitative reverse transcriptase-polymerase chain reaction techniques during different stages of the estrus cycle. The results confirm that an increase in FGF-beta mRNA levels occurs over the course of the estrus cycle. No FGF-beta gene expression was detected during diestrus, the non-hormonal phase of the cycle, or at the early proestrus stage of the cycle. During late proestrus and estrus, FGF-beta mRNA was predominantly localized to granulosa cells of the dominant follicles, and to a lesser extent, to secondary antral follicles not committed to ovulation. These cells also expressed FGF-beta mRNA during this phase of follicular development, albeit in low abundance. During metestrus, after ovulation, in the newly formed corpora lutea FGF-beta mRNA levels were maximal, however on entering the next cycle commencing at diestrus, no FGF-beta mRNA was observed in the degenerating corpora lutea. These results indicate that expression of the FGF-beta gene is differentially regulated during the estrus cycle. The biological significance of this expression and the potential role of FGF-beta in local intra-ovarian regulation of the repetitive cycles of follicular differentiation, proliferation and maturation associated with ovarian revascularization are discussed.

Animals↗
PubMed2004

[Detection of ASPL-TFE3 fusion gene by reverse transcriptase polymerase chain reaction in paraffin-embedded tumor tissues of alveolar soft part sarcoma].

OBJECTIVE: To investigate the significance of detecting chimeric mRNA resulting from t(X;17)(p11.2;q25) in paraffin-embedded tumor tissues of alveolar soft part sarcoma (ASPS). METHODS: Formalin-fixed, paraffin-embedded tumor tissues from 8 cases of alveolar soft part sarcoma and 15 cases of controls (including 6 alveolar rhabdomyosarcomas, 6 renal cell carcinomas, 2 paragangliomas and 1 granular cell myoblastoma) were retrieved from the archival materials. ASPL-TFE3 fusion transcripts were analyzed in all samples by reverse transcriptase-polymerase chain reaction (RT-PCR). The quality of the mRNA was assessed using the house-keeping gene beta-actin. RESULTS: ASPL-TFE3 fusion transcripts were detected in 6 of the 8 ASPS cases (4 being type 2 and 2 being type 1). The remaining 2 cases were negative for both beta-actin and ASPL-TFE3. No ASPL-TFE3 mRNA expression was detected in all the controls. PAX3/7-FKHR fusion transcripts were also detected in 4 of the 6 alveolar rhabdomyosarcoma samples. CONCLUSIONS: The expression of ASPL-TFE3 fusion transcripts in paraffin-embedded tumor tissues can serve as an useful molecular marker in the diagnosis of ASPS. It may also be helpful in elucidating the underlying pathogenesis of ASPS in subsequent retrospective studies.

Adolescent↗
PubMed2001

Reverse-transcriptase polymerase chain reaction of the maspin gene in the detection of bone marrow breast carcinoma cell contamination.

BACKGROUND: Maspin is a molecular marker used for the detection of contaminating breast carcinoma (BC) cells in peripheral blood and lymph nodes. However, its specificity has been questioned recently. The objective of this study was to verify the specificity of this marker and to determine the incidence of positive bone marrow results in patients with BC who are eligible for high-dose chemotherapy (HDT) both in early and advanced disease stages and before and after treatment. METHODS: Bone marrow specimens from 41 patients with BC as well as from 35 normal volunteers and 17 patients with hematologic tumors were examined for maspin transcript expression by a modified nested reverse transcriptase-polymerase chain reaction technique. RESULTS: Maspin transcript was found in all normal and neoplastic breast tissues and in none of the 35 normal bone marrow specimens (specificity, 100%; 95% confidence interval, 90-100%). However, the transcript was found in 40% of the bone marrow samples from patients with hematologic malignancies. Thus, this marker appears very specific for discriminating between normal controls and patients with BC, but it cannot be considered disease specific. Among patients with BC, bone marrow was positive for the maspin transcript in 32% of patients with early-stage disease and in 75% of patients with metastatic disease before chemotherapy. After treatment, in 75% of patients with early-stage disease and in 50% of patients with metastatic disease, the bone marrow results became maspin negative. CONCLUSIONS: On the basis of the current data, although it is not disease specific, maspin is a reliable marker for detecting bone marrow molecular disease in patients with BC and should be considered for prospective studies as a prognostic indicator and as an assay for monitoring residual disease.

Biomarkers, Tumor↗
PubMed2000

In situ reverse-transcriptase polymerase chain reaction demonstration of the EWS/FLI-1 fusion transcript in Ewing's sarcomas and peripheral primitive neuroectodermal tumors.

It is now widely accepted that the EWS/FLI-1 fusion transcript is associated with tumors of the Ewing family. To test whether it is possible to detect the fusion transcript by means of combining polymerase chain reaction (PCR) methodology and immunohistochemistry, we investigated tumors of the Ewing family using in situ reverse transcriptase (RT)-PCR. We were able to demonstrate the t(11;22) fusion transcript in five of six cases of Ewing's sarcoma and four of four peripheral primitive neuroectodermal tumors. These results were confirmed using fluorescence in situ hybridization in seven tumor samples. In situ RT-PCR-labeled fusion transcripts were found in virtually all tumor cells within a given sample, indicating that each cell possessed the t(11;22) transcript. We conclude from these results that in situ RT-PCR can be used for the rapid detection of EWS/FLI-1 fusion transcripts in biopsy material. The findings also suggest that all cells of the tumors of the Ewing family carry the EWS/FLI-1 fusion transcript.

Adolescent↗
PubMed1999

Comparison of specific and random priming in the reverse transcriptase polymerase chain reaction for genotyping group A rotaviruses.

This study describes an approach to the molecular typing of rotaviruses which requires only a single RNA extraction and reverse transcription (RT) reaction using random primers. Random-primed RT provides complementary DNA (cDNA) which can be used not only for G- and P-typing polymerase chain reactions (PCR), but also for the detection of other RNA viruses which may act as enteric pathogens. It is a sensitive and specific method that can detect 10 virus particles/ml of 10% faecal suspension provided the cDNA is amplified in a nested typing-PCR. Of 121 specimens positive for rotavirus by EM and analysed using this method, only 8% could not be G- or P-genotyped. The untyped samples were tested again performing the RT reaction with G- and P-specific primers, achieving a 5% increase in sensitivity. Comparing G-genotyping against G-serotyping, 92% were genotyped through random priming RT-PCR whereas only 64% were serotyped using G-serotype specific monoclonal antibodies (MAbs).

Aged↗
PubMed1999

Molecular diagnosis of dengue by reverse transcriptase-polymerase chain reaction.

The techniques of reverse transcriptase-polymerase chain reaction (RT-PCR) and subsequent PCR were employed in the analysis of serum samples from a range of patients from the Caribbean Epidemiology Centre (CAREC) member countries. Results were compared with those from viral isolation and immunofluorescence. In the second part of the study, ten serum samples were stored for one week under four sets of conditions: -20 degrees C, 4 degrees C, 25 degrees C, and thawed (20 degrees C) and frozen (-20 degrees C) daily. After one week of each treatment the samples were analysed by RT-PCR and PCR. 90.4% of results from PCR agreed with results from viral isolation (VI) and fluorescent antibody (FA) detection. All PCR positive samples originated from sera collected within five days of the date of onset of fever. Frozen, refrigerated and repeat freeze-thawed samples gave consistent positive results by RT-PCR. After storage at 25 degrees C, however, half the dengue-positive samples were negative by RT-PCR. The results indicate the sensitivity and reliability of this rapid technique and its applicability in the Caribbean. It provides a preliminary assessment of its advantages and limitations under certain conditions of serum collection and storage.

Dengue↗