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Results for “Reverse Transcriptase Polymerase Chain Reaction”

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At least 127 records · Page 7Linked to original sources
PubMed2000

Monitoring of minimal residual disease in patients with MLL-AF6-positive acute myeloid leukaemia by reverse transcriptase polymerase chain reaction.

We studied 210 unselected patients with acute myeloid leukaemia (AML) for MLL abnormalities. Twenty-seven patients (13%) with rearranged MLL genes were identified by means of Southern blot analysis. An MLL-AF6 fusion transcript was detected in six patients by a reverse transcriptase polymerase chain reaction (RT-PCR) for the MLL-AF6 translocation. Sequence analysis showed fusion of MLL exon 7 as well as exon 6 (two patients) or MLL exon 6 as well as exon 5 (four patients) to AF6 exon 2. In only three patients could the t(6;11) also be identified by cytogenetic and/or fluorescence in situ hybridization (FISH) analysis. The MLL-AF6-positive patients were monitored by RT-PCR for a period of 6-33 months. Complete haematological remission (CR) was achieved in all six cases, but was short in 5/6 patients (range 2.6-8.3 months). In these five patients, the MLL-AF6 transcripts were detected in every sample tested after induction and consolidation chemotherapy. One patient received autologous bone marrow transplantation (BMT) which also did not lead to PCR negativity. Intensive salvage therapy was unable to induce a second remission in the relapsed patients. One of the six MLL-AF6-positive patients achieved a molecular CR. He is still in CR at 33 months after diagnosis. Survival analysis indicates a poor prognosis in MLL-AF6-positive patients. The median event-free survival was 6.8 months, the median overall survival 15 months. Persistent PCR positivity was consistently associated with relapse. Thus, RT-PCR provides a valuable and sensitive tool for the identification of t(6;11)-positive AML and the monitoring of response to treatment in these patients. The results of RT-PCR may be useful to evaluate therapeutic procedures and to make treatment decisions, which will enable molecular remissions to be achieved and improve the clinical outcome in this group of patients.

Acute Disease↗
PubMed2000

Detection of circulating tumor cells by reverse transcriptase polymerase chain reaction of maspin in patients with breast cancer undergoing conventional-dose chemotherapy.

PURPOSE: To establish, in patients with breast cancer subjected to primary conventional chemotherapy and enrolled in a prospective study, the mobilizing effect of therapy on potentially neoplastic cells by means of a reverse transcriptase polymerase chain reaction (RT-PCR) assay for mRNA of maspin, a protein related to the serpin family of protease inhibitors. PATIENTS AND METHODS: Peripheral-blood samples were collected from 30 patients with histologically proven breast cancer before and 4 and 8 days after conventional chemotherapy for three consecutive courses. A total of 216 samples were screened for the presence of maspin mRNA by RT-PCR. RESULTS: Before therapy, all samples but one were negative. After chemotherapy, 11 patients (38%) had positive samples. No difference in the rate of positivity was observed between groups defined according to initial stage, type of chemotherapy, Ki-67-related proliferative activity, or CA 15.3 expression. CONCLUSION: Our results confirm that RT-PCR for maspin mRNA is a sensitive assay for the study of circulating potentially neoplastic mammary cells in patients with breast cancer. Moreover, our findings indicate a marked effect of conventional-dose chemotherapy on the mobilization of these cells in breast tumors. In our series of patients, this phenomenon does not seem to be associated with other known risk factors. Finally, the data suggest, without proving, an association between the presence of circulating maspin positive cells and a higher risk of disease progression. If this association could be confirmed, then the assay could have prognostic significance. However, larger confirmatory studies are necessary.

Adult↗
PubMed2002

Comparative detection of lymph node micrometastases of stage II colorectal cancer by reverse transcriptase polymerase chain reaction and immunohistochemistry.

PURPOSE: Inconsistent conclusions have been drawn about the clinical significance of micrometastases in lymph nodes (LNs) of node-negative colorectal cancer (CRC) patients. We performed a comparative study of detection of micrometastases using immunohistochemistry (IHC) by anti-cytokeratin antibody and carcinoembryonic antigen (CEA)-specific reverse-transcriptase polymerase chain reaction (RT-PCR) in the same patients, in an attempt to move closer to their clinical application. PATIENTS AND METHODS: Sixty-four CRC patients, with RNA of good quality available from paraffin-embedded LN specimens, were selected from 84 stage II patients who underwent curative surgery between 1988 and 1996. We investigated associations between the presence of micrometastases by each method and prognosis. RESULTS: Micrometastases were detected in 19 (29.6%) of 64 patients by RT-PCR and in 35 (54.7%) of 64 patients by IHC. By RT-PCR analysis, patients exhibiting a positive band for CEA mRNA had a significantly worse prognosis than those who were RT-PCR-negative, with respect to both disease-free and overall survival (P =.027 and.015, respectively). By IHC analysis, the presence of micrometastasis did not predict patient outcome in terms of either disease-free or overall survival. Infiltrating pattern of tumor growth characteristic was significantly associated with shorter disease-free survival among various clinical or pathologic factors. By multivariate Cox regression analysis, micrometastasis detected by RT-PCR and the Crohn's-like lymphoid reaction were both independent prognostic factors. CONCLUSION: Micrometastases detected by RT-PCR, but not IHC, may be of clinical value in identifying patients who may be at high risk for recurrence of CRC and who are therefore likely to benefit from systemic adjuvant therapy.

Adenocarcinoma↗
PubMed2000

Detection of micrometastatic prostate cancer cells in the lymph nodes by reverse transcriptase polymerase chain reaction is predictive of biochemical recurrence in pathological stage T2 prostate cancer.

PURPOSE: We evaluated whether detecting prostate cancer cells by the nested reverse transcriptase-polymerase chain reaction (RT-PCR) in lymph nodes has predictive value for serum prostate specific antigen (PSA) recurrence in patients undergoing radical prostatectomy. MATERIALS AND METHODS: We assessed the presence of prostate cancer cells by RT-PCR for prostate specific membrane antigen and PSA assay in lymph nodes dissected from 38 patients with localized prostate cancer treated with radical prostatectomy. The results of nested RT-PCR assay were compared with biochemical recurrence. RESULTS: Nested RT-PCR was positive in the lymph nodes of 2 of 18 patients (11%) with stage pT2a and 5 of 20 (25%) with stage pT2b disease. All 7 patients had biochemical recurrence. We noted a significant difference in the Kaplan-Meier recurrence-free actuarial probability curve in those with positive and negative nested RT-PCR results for prostate specific membrane antigen, PSA and prostate specific membrane antigen-PSA in the lymph nodes (p = 3.02x10(-7), 2.23x10(-7) and 3.02x10(-7), respectively). Multivariate analysis of serum PSA, Gleason score and preoperative RT-PCR assay in peripheral blood showed that nested RT-PCR for prostate specific membrane antigen, PSA and prostate specific membrane antigen-PSA in the lymph nodes were independent predictors of recurrence (p = 0.0089, 0.0075 and 0.0089, respectively). CONCLUSIONS: Nested RT-PCR of the lymph nodes may be a useful pretreatment prognostic test for patients undergoing radical prostatectomy. Further research is necessary using a much larger number of patients with a longer followup.

Aged↗
PubMed1998

Competitive CBFbeta/MYH11 reverse-transcriptase polymerase chain reaction for quantitative assessment of minimal residual disease during postremission therapy in acute myeloid leukemia with inversion(16): a pilot study.

PURPOSE: (1) Quantification of minimal residual disease (MRD) by competitive CBFbeta/MYH11 reverse-transcriptase polymerase chain reaction (RT-PCR) in patients with acute myeloid leukemia (AML) and inversion(16) [inv(16)] during postremission therapy, (2) comparison of this method with conventional two-step RT-PCR, and (3) evaluation of a potential prognostic value. PATIENTS AND METHODS: MRD of six consecutive adult patients with AML and inv(16)(p13;q22) or t(16;16)(p13;q22) who entered complete remission (CR) was monitored by competitive CBFbeta/MYH11 RT-PCR in their bone marrow (BM) during postremission therapy with high-dose cytarabine (HiDAC) or after BM transplantation with a matched unrelated-donor marrow (MUD-BMT) during an observation period of 4.5 to 27 months after initiation of treatment. RESULTS: Competitive PCR showed a gradual decline by at least 4 orders of magnitude after 7 to 9 months in patients in continuous CR (CCR), while one patient who relapsed after 13.5 months only achieved a reduction by 2 orders of magnitude at the end of consolidation therapy. A rapid decrease below the detection limit was observed within 1 month in two patients after MUD-BMT. A temporary reappearance of molecular MRD was observed in these patients during immunosuppression for graft-versus-host disease (GvHD). After reduction of immunosuppression, the level of MRD dropped again below the PCR detection limit. Molecular monitoring by conventional two-step RT-PCR yielded comparable results only when multiple assays per time point were performed, while single-assay RT-PCR gave misleading results. CONCLUSION: Competitive RT-PCR is a valuable tool for molecular monitoring during postremission chemotherapy, as well as after BMT.

Adolescent↗
PubMed2005

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

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), all lesions became positive for tyrosinase mRNA. CONCLUSION: Metabolically inactive or dormant melanocytes are probably present within vitiligo areas, and may be activated by exogenous or endogenous stimuli.

Adolescent↗
PubMed1999

Detection of genomic- and minus-strand of hepatitis C virus RNA in the liver of chronic hepatitis C patients by strand-specific semiquantitative reverse-transcriptase polymerase chain reaction.

Studies aimed at correlating the intrahepatic hepatitis C virus (HCV)-RNA level and anatomo-clinical features have been difficult because of sensitivity and specificity shortcomings of available techniques. We titered the genomic- and minus-strand HCV RNAs by a strand-specific, semiquantitative, genotype-independent reverse-transcriptase polymerase chain reaction (RT-PCR) in the liver tissue of 61 patients with chronic hepatitis C. Findings were correlated with the levels of HCV RNA in the serum, the HCV genotype, the expression of intrahepatic HCV antigens, the histological activity (using separate scores for the lobular and the portal/periportal necroinflammatory activity and for the fibrosis), and the response to interferon alfa (IFN-alpha) treatment. Genomic- and minus-strand HCV RNA were detected in 59 and 57 liver specimens, respectively. The HCV-RNA level in the serum correlated with the genomic-strand, but not with the minus-strand, HCV-RNA titer in the liver. No correlations were found between either strand of the intrahepatic HCV RNA and the level of expression of HCV antigens in the liver, or with the grading/staging of the underlying liver disease. The response to IFN-alpha treatment could be predicted by the serum HCV-RNA level and genotype, but not by the intrahepatic level of genomic- or minus-strand HCV RNA. These results suggest that, although the detection of the minus-strand HCV RNA reliably identifies the presence of replicating HCV in its target organ, the quantitative measurement of viremia remains the clinically meaningful "golden standard" for assessing the level of HCV replication.

Adult↗
PubMed2003

Quantitation of GD2 synthase mRNA by real-time reverse transcriptase polymerase chain reaction: clinical utility in evaluating adjuvant therapy in neuroblastoma.

PURPOSE: Minimal residual disease (MRD) is one of the final hurdles to cancer cure. Because therapy (myeloablation, immunotherapy, or differentiation) for MRD is applied at the time of clinical remission, objective surrogate markers are needed to gauge treatment efficacy. PATIENTS AND METHODS: Using quantitative reverse transcriptase polymerase chain reaction (RT-PCR) of GD2 synthase (beta1,4-N-acetylgalactosaminyltransferase, EC 2.4.1.92) mRNA, we evaluated MRD response to anti-GD2 monoclonal antibody 3F8 adjuvant therapy, namely, one cycle of radioimmunotherapy using iodine-131 ((131)I)-3F8 plus one cycle of unlabeled 3F8 in 45 stage 4 neuroblastoma patients (newly diagnosed or without prior relapse) on the N7 protocol at Memorial Sloan-Kettering Cancer Center. The prognostic effect of MRD in their bone marrows before and after this phase of adjuvant therapy on progression-free survival (PFS) and overall survival (OS) was also analyzed. RESULTS: Before 3F8 treatment, 24 of 45 patients were in complete remission (CR), 12 were in very good partial remission (VGPR), and nine were in partial remission (PR), according to criteria from International Neuroblastoma Staging System plus (131)I-3F8 scan; 71% had detectable tumor cells in marrow by real-time RT-PCR. Of the 32 positive patients, 20 became negative after therapy, with a 63% efficacy. When patients were stratified by CR/VGPR versus PR, GD2 synthase positivity was prognostic when detected before 3F8-targeted therapy (PFS, P =.045 and OS, P =.010). Persistent marker positivity was also predictive of PFS (P =.035) and OS (P =.027). Patients who succumbed to the disease had transcript levels four times higher than those who remain alive. CONCLUSION: GD2 synthase mRNA is a useful surrogate marker for evaluating adjuvant treatment efficacy in neuroblastoma with prognostic potential.

Adult↗
PubMed1994

Sensitive detection of occult breast cancer by the reverse-transcriptase polymerase chain reaction.

PURPOSE: Detection of occult carcinoma in patients with breast cancer may aid the establishment of prognosis and development of new therapeutic approaches. To improve on existing methods of detection, we have developed a reverse-transcriptase polymerase chain reaction (RT-PCR) assay for keratin 19 (K19) transcripts to identify mammary carcinoma cells in the peripheral blood and bone marrow of patients with breast cancer. PATIENTS AND METHODS: Peripheral-blood or bone marrow samples obtained from 34 patients with stages I to IV breast cancer and 39 control subjects without breast cancer were screened for K19 mRNA by nested primer PCR. RESULTS: In reconstitution experiments, K19 RT-PCR reliably detected 10 mammary carcinoma cells in 1 million normal peripheral-blood mononuclear (PBMN) cells. Four of 19 patients with stage IV breast cancer had detectable K19 transcript in peripheral blood. Five of six patients with histologically negative bone marrow biopsies following preablative chemotherapy and before autologous bone marrow transplant (BMT) were positive by this assay. Stem-cell apheresis harvests obtained from one of these patients and three additional patients immediately before BMT were all K19-negative. K19 RT-PCR analysis of CSF from a breast cancer patient with known carcinomatous meningitis was also positive. Thirty-eight of 39 non-breast cancer patients had negative K19 RT-PCR assays. The one exception was a patient with chronic myelogenous leukemia. CONCLUSION: RT-PCR of K19 is a sensitive, specific, and rapid method for detection of occult mammary carcinoma cells in the peripheral blood and bone marrow of patients with breast cancer. The presence of residual breast cancer cells in histologically normal bone marrow aspirates but not in stem-cell apheresis harvests is a frequent finding. This assay may be useful in diagnosing metastatic disease, as well as in monitoring the effectiveness of systemic therapy.

Base Sequence↗
PubMed1998

Limitations of specific reverse-transcriptase polymerase chain reaction markers in the detection of metastases in the lymph nodes and blood of breast cancer patients.

PURPOSE: This study was performed to evaluate the potential of specific mRNA markers to detect micrometastases by reverse-transcriptase polymerase chain reaction (RT-PCR) and Southern blot analysis of sentinel lymph nodes (SNs) and blood from patients with breast cancer. PATIENTS AND METHODS: We assessed the specificity of carcinoembryonic antigen (CEA), cytokeratin-19 (CK-19), CK-20, gastrointestinal tumor-associated antigen-733.2 (GA733.2), and mucin-1 (MUC-1) in the blood of healthy donors (n = 13) and lymph nodes from patients without cancer (n = 3) by RT-PCR assay. The sensitivity of the RT-PCR assay for the target mRNA markers was assessed in breast cancer cell lines (n = 4), primary breast tumors (n = 8), and the frozen sections of SNs (n = 22) from 22 patients with American Joint Committee on Cancer (AJCC) stages I to IIIA breast cancer. RESULTS: CK-20 was the only mRNA marker not detected in lymph nodes or blood from patients without cancer. Both the blood and lymph nodes from patients without cancer expressed CEA, CK-19, GA733.2, and MUC-1 mRNA. All four breast cancer cell lines and six of eight primary breast tumors expressed all five mRNA markers. Expression of mRNA by the RT-PCR assay in the frozen-section SNs (n = 12) without metastases by conventional histopathology ranged from 8% (CK-20) to 92% (GA733.2). Detection of RT-PCR cDNA products in frozen-section SNs was increased with Southern blot analysis compared with ethidium bromide gel electrophoresis (EtBr) for all mRNA markers except CK-19. CONCLUSION: CEA, CK-19, GA733.2, and MUC-1 show no diagnostic value as mRNA markers for the detection of micrometastases by the RT-PCR assay because they are expressed in the blood and lymph nodes of patients without cancer. Further studies are needed to assess the sensitivity of CK-20 to detect micrometastases by the RT-PCR assay in the blood and frozen-section SNs of patients with breast cancer.

Antigens, Neoplasm↗
PubMed2002

Comparison of vero cell plaque assay, TaqMan reverse transcriptase polymerase chain reaction RNA assay, and VecTest antigen assay for detection of West Nile virus in field-collected mosquitoes.

Mosquitoes collected during the epidemic of West Nile virus (WN) in Staten Island, NY, during 2000 were identified to species, grouped into pools of up to 50 individuals, and tested for the presence of WN by using TaqMan reverse transcriptase polymerase chain reaction (RT-PCR) to detect West Nile viral RNA, Vero cell plaque assay to detect infectious virus, and VecTest WNV/SLE Antigen Panel Assay. A total of 10,866 specimens was tested in 801 pools. Analysis of results indicated that TaqMan RT-PCR detected 34 WN-positive pools, more than either of the other techniques. The plaque assay detected 74% of the pools positive by TaqMan, and VecTest detected 60% of the pools positive by TaqMan. The VecTest assay detected evidence of West Nile viral antigen in 67% of the pools that contained live virus detected by plaque assay. A WN enzyme immunoassay performed similarly to the VecTest WN assay. Differences in performance were related to relative sensitivity of the tests. Infection rates of WN in Culex pipiens and Cx. salinarius calculated by the 3 techniques varied, but each estimate indicated a high infection rate in the population. Positive and negative attributes of each procedure, which may influence how and where they are used in surveillance programs, are discussed.

Aedes↗
PubMed2002

Quantitative assessment of gene expression in highly purified hematopoietic cells using real-time reverse transcriptase polymerase chain reaction.

OBJECTIVE: Quantitative assessment of gene expression in stem cells is essential for understanding the molecular events underlying normal and malignant hematopoiesis. The aim of the present study was to develop a method for precise quantitation of gene expression in small subsets of highly purified CD34(+)CD38(-) stem cell populations. MATERIALS AND METHODS: Real-time quantitative reverse transcriptase polymerase chain reaction (RT-PCR) was used to quantitate housekeeping and drug resistance gene expression in cDNA obtained from 300 CD34(+)CD38(-) cells without cDNA amplification or nested PCR techniques. RESULTS: Validation experiments in cell lines showed efficient, representative and reproducible gene amplification using 300-cell real-time quantitative RT-PCR. Sensitivity was confirmed in dilutional experiments and by detection of the low-copy gene PBGD. GAPDH was found to be a useful reference gene in normal and leukemic CD34(+)CD38(-) cells. In contrast, 18S rRNA content varied 100-fold to 1000-fold in these populations. Moreover, expression of 18S rRNA was significantly lower in leukemic CD34(+)CD38(+) cells compared to normal CD34(+)CD38(+) cells (p = 0.002). Expression of MDR-1 (18-fold, p < 0.0005), MRP-1 (3.8-fold, p < 0.05), and LRP (1.8-fold, NS) was higher in normal CD34(+)CD38(-) compared to CD34(+)CD38(+) cells. CONCLUSIONS: Real-time quantitative RT-PCR is a valuable tool for precise quantitation of gene expression in small subsets of hematopoietic cells. Using this method, we showed the inappropriateness of 18S as a reference gene in these progenitors and the down-regulation of drug-resistance-related genes early in hematopoiesis.

ADP-ribosyl Cyclase↗
PubMed2003

Determination of prostate specific antigen mRNA in peripheral blood by reverse transcriptase polymerase chain reaction and a simple chemiluminometric hybridization assay in a high-throughput format.

In recent years, the mRNA for prostate-specific antigen (PSA) has been investigated as a potential marker for molecular staging of prostate cancer. We report a simple, rapid, and sensitive assay protocol for the quantification of PSA mRNA in peripheral blood by using reverse transcriptase polymerase chain reaction (RT-PCR) and a chemiluminometric hybridization assay. A recombinant RNA internal standard (IS) that has the same size and primer binding sites as the PSA mRNA is included in the RT-PCR mixture. Total RNA from the sample is coextracted with a constant amount of IS RNA and subjected to RT-PCR. Amplified sequences are labeled with biotin during PCR by using a biotinylated upstream primer. The products are heat-denatured and hybridized with oligonucleotide-specific probes (for PSA and IS) that are immobilized in microtiter wells. Immobilization of oligonucleotide probes is achieved by adsorption of their conjugates with bovine serum albumin. The hybrids are measured using alkaline phosphatase-labeled streptavidin and a dioxetane chemiluminogenic substrate. The ratio of the luminescence values obtained for the PSA mRNA and the RNA IS is a linear function of the initial amount of PSA mRNA present in the sample prior to RT-PCR amplification. The linear range extended from 50 to 500,000 PSA mRNA copies, and the overall reproducibility of the assay, including RT-PCR and hybridization, ranged from 11.5 to 14.2%. Samples containing total RNA from PSA-expressing LNCaP cells give luminescence ratios that are linearly related to the number of cells in the range of 0.04-400 cells. The method was applied to PSA mRNA determination in peripheral blood of healthy individuals, patients with benign prostate hyperplasia, patients with prostate cancer, and patients with other types of localized cancer.

Female↗
PubMed2003

Lymphotoxin beta expression is high in chronic lymphocytic leukemia but low in small lymphocytic lymphoma: a quantitative real-time reverse transcriptase polymerase chain reaction analysis.

BACKGROUND AND OBJECTIVES: The lymphotoxin beta (LTB) gene, localized to the major histocompatibility complex region on chromosome 6p21.3, has an important role in the formation of germinal center reactions and regulation of immune response and apoptosis. Our aim was to determine LTB gene expression in different hematologic neoplasias. DESIGN AND METHODS: We determined the expression of LTB using quantitative real-time reverse transcriptase polymerase chain reaction (RT-PCR) on a series of RNA samples from CD3(+) T cells and CD19(+) B cells isolated from peripheral blood (n=7); CD19(+) B cells isolated from lymph nodes (n=11) and from patients with acute lymphoblastic leukemia (ALL; n=16), acute myeloid leukemia (AML; n=43), chronic myeloid leukemia (CML; n=12), mantle cell lymphoma (MCL; n=19), chronic lymphocytic leukemia (CLL; n=32) and small lymphocytic lymphoma (SLL; n=22). RESULTS: The expression level of LTB in CD3(+) T cells and CD19(+) B cells isolated from blood was ten to forty times lower than that in normal CD19(+) B cells isolated from lymph nodes. In malignant myeloid cells the expression levels were very low, whereas in malignant lymphoid cells the expression was higher than in myeloid cells, being highest in MCL and CLL (20.2+/-14.0 ng/microL and 81.0+/-116.3 ng/microL) and low in SLL (4.5+/-3.6 ng/microL; p=0.001). We did not find correlations between LTB expression and hematoclinical parameters (risk groups, immunophenotypes and overall survival). INTERPRETATION AND CONCLUSIONS: A distinct difference in expression of LTB in CLL and SLL indicates that these morphologically similar B-cell malignancies are molecularly different. Further studies are needed to investigate the prognostic value of LTB and any role that LTB may have in determining whether the malignant B cells manifest a leukemia or lymphoma.

Adult↗
PubMed2000

Characterization of the capsid protein gene from a nodavirus strain affecting the Atlantic halibut Hippoglossus hippoglossus and design of an optimal reverse-transcriptase polymerase chain reaction (RT-PCR) detection assay.

A 1349 nucleotide fragment of the RNA2 from a nodavirus affecting Atlantic halibut Hippoglossus hippoglossus was characterised and the nuclotide sequence (accession no. AJ245641) was employed to develop an optimal reverse-transcriptase polymerase chain reaction (RT-PCR) detection assay. The sequenced part of the RNA2 of Atlantic halibut nodavirus (strain AH95NorA) was highly similar in organisation to that of the RNA2 of striped jack nervous necrosis virus (SJNNV), and comprised features common to all nodaviruses. These characteristics confirmed that the virus that causes viral encephalopathy and retinopathy (VER) in Atlantic halibut is a nodavirus. The nucleotide sequence of the 1349 nucleotide fragment of Atlantic halibut nodavirus RNA2 was 80% identical to the RNA2 of SJNNV. The T2 region (830 nucleotides) of the RNA2 of Atlantic halibut nodavirus shared 98% of the nucleotide sequence when compared with the homologous region of barfin flounder nervous necrosis virus (BFNNV), while the nucleotide sequence identity to SJNNV in this region was 76%. Phylogenetic analysis based on the nucleotide sequences of the T4 region (421 nucleotides) of Atlantic halibut nodavirus and of other fish nodaviruses revealed a close relationship to the nodaviruses of the barfin flounder clad that have been found in other cold-water species (Pacific cod Gadus macrocephalus and barfin founder Verasper moseri). The nucleotide sequence of the RNA2 of Atlantic halibut nodavirus included some features that differ from that of SJNNV. The ORF of the RNA2 of Atlantic halibut nodavirus lacked 6 nucleotides through a single deletion and a 5-nucleotide deletion, separated by 4 nucleotides. The 3'-non-encoding region contained a 21 nucleotide insert and a 3 nucleotide deletion when compared with SJNNV. In comparison with the RNA2 of SJNNV, the 3'-non-encoding region showed a nucleotide sequence identity of 84.5%. A primer set based on the Atlantic halibut nodavirus nucleotide sequence was employed in order to design an optimal RT-PCR. The detection limit of the PCR was 10 to 100 copies of plasmid, while the detection limit of the RT-PCR assay was 100 to 1000 copies of in vitro transcribed viral RNA.

Animals↗
PubMed1995

Immunomagnetic purification of human breast carcinoma cells allows tumor-specific detection of multidrug resistance gene 1-mRNA by reverse transcriptase polymerase chain reaction in fine-needle aspirates.

BACKGROUND: The heterogenous composition of tumors is a major obstacle for the measurement of mRNA levels in cancer cells. We report here a combination of immunomagnetic purification of cancer cells and reverse transcriptase polymerase chain reaction (RT-PCR) that enables highly sensitive detection of multidrug resistance gene 1 (MDR1)-mRNA levels in human breast carcinoma cells obtained from fine needle aspirates (FNA). EXPERIMENTAL DESIGN: Murine mAb 115D8 directed against episialin (MUC1/MAM6, epithelial membrane Ag) was used in combination with goat anti-mouse-coated magnetic microbeads to purify human T47D breast carcinoma cells (115D8+, MDR1-) from different mixtures with COLO320 human colon carcinoma cells (115D8-, MDR1+) and to purify carcinoma cells from FNA taken from axillary lymph node metastases in breast cancer patients. The efficacy of the purification was determined by FACS-analysis and by measurement of MDR1-mRNA levels by semiquantitative RT-PCR. RESULTS: FACS-analysis demonstrated that T47D cells could be purified up to 99.8% from mixtures with COLO320 cells ranging from 3:1 to 1:3. The MDR1-mRNA level in these enriched mixtures, as detected by RT-PCR, was reduced 250-fold. It was demonstrated that MDR1 expression present in an FNA from a lymph node metastasis of breast carcinoma could be attributed completely to the leukocytes present in this FNA, because MDR1 expression was no longer detectable after purification of the tumor cells. CONCLUSION: The combination of immunomagnetic purification of breast carcinoma cells and RT-PCR enables the measurement of cancer-specific MDR1 mRNA levels in small cell samples obtained by FNA.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed2002

Transcript abundance in mouse pituitaries with altered growth hormone expression quantified by reverse transcriptase polymerase chain reaction implicates transcription factor Zn-16 in gene regulation in vivo.

The correlation of growth hormone (GH) mRNA abundance and expression of specific transcription factors was studied in pituitaries of panhypopituitary (Ames df/df and Snell dwJ/dwJ dwarf), isolated GH-deficient (lit/lit), and GH-overproducing (growth hormone-releasing hormone [GHRH] transgenic) mice compared with normal littermates. A fluorescence-based reverse transcriptase polymerase chain reaction assay was developed for seven target mRNAs: GH, prolactin (PRL), pro-opiomelanocortin (POMC), alpha-subunit of the glycoprotein hormones (alphaSU), Pit-1, Prop-1, and Zn-16. Amplification parameters for each of these primer pairs were determined in order to calculate initial mRNA transcript number. The reproducibility of the assay was found to be +/-10% for either Pit-1 or Zn-16 mRNAs measured in characterized murine GHFT1-5 somatotroph precursor cells. The cell extracts also showed an increased abundance of both Zn-16 and Pit-1 mRNAs when compared with whole pituitary extracts. Measurement of copy number in normal pituitaries showed that for every 10(6) GH or PRL mRNAs, there were 3 x 10(5) POMC, 4 x 10(4) alphaSU, 2 x 10(3) Pit-1, and only 70 Zn-16 or Prop-1 transcripts. Transcript abundance in GH-altered mice as a percentage of copy number per normal gland showed that POMC was significantly reduced in dwJ/dwJ (p < 0.01) and df/df (p < 0.05) mice. AlphaSU mRNA was reduced in df/df (p < 0.05), dwJ/dwJ (p < 0.05), and lit/lit (p < 0.05) mice, but not in GHRH-excess mice. PRL mRNA was not detected in dwarf mice, reduced to 52% of normal in lit/lit (p < 0.05), and unchanged in GHRH-excess animals. GH mRNA was not detected in dwarf mice, reduced to 1.3% in lit/lit (p < 0.005), and increased to 242% in GHRH-excess mice (p < 0.05). Pit-1 mRNA was not detected in dwarf mice, was 2.9% of normal in lit/lit (p < 0.005) mice, and increased to 200% in GHRH-excess mice (p < 0.05). Prop-1 was not present in dwarf mice, was decreased to 1.4% in lit/lit (p < 0.01), and increased to 223% in GHRH-excess mice (p < 0.05). Zn-16 abundance in df/df mice was significantly reduced (p < 0.05) to 4.8% of normals, to 6.3% of normals in dwJ/dwJ (p < 0.005), to 6.1% of normals in lit/lit (p < 0.005) mice, and significantly elevated in GHRH-excess mice to 197% (p < 0.05). Altered pituitary mRNA abundance was found for several products not previously measured, or thought not to be affected by these mutations. Correlation of GH mRNA abundance with transcription factor copy number showed a significant correlation for Pit-1, Prop-1, and Zn-16. These quantitative analyses provide the first in vivo evidence that Zn-16 mRNA abundance correlates with GH expression.

Animals↗
PubMed2001

Detection of occult melanoma cells in paraffin-embedded histologically negative sentinel lymph nodes using a reverse transcriptase polymerase chain reaction assay.

PURPOSE: Detection of occult metastasis before the development of clinical disease could allow more accurate staging, appropriate follow-up procedures, and adjuvant therapies in patients with malignant melanoma (MM). The sentinel lymph node (SLN) has been proposed as a reliable predictor of metastatic disease in the lymphatic basin draining the primary melanoma. In this study, we screened both paraffin-embedded SLNs and peripheral-blood (PB) samples from MM patients at various stage of disease using a multimarker reverse transcriptase polymerase chain reaction (RT-PCR) assay. The prognostic significance of the presence of PCR-positive markers was also evaluated. PATIENTS AND METHODS: Total RNA was obtained from paraffin-embedded SLN sections and PB samples of 75 MM patients. RT-PCR was performed using tyrosinase and MelanA/MART1 as melanoma-associated markers. Radiolabeled PCR products were analyzed on denaturing polyacrylamide gels. RESULTS: Good sensitivity of the RT-PCR assay on archival tissues was demonstrated after comparison of RT-PCR results on frozen and paraffin-embedded SLNs from 16 MM patients. Significant correlation between the disease stage and marker expression in both PB and SLN samples was observed; the highest value was for patients who were positive for both markers in SLN (P =.006). Progression of disease was significantly associated with the total number of PCR-positive markers in both PB (P =.034) and SLN (P =.001) samples. CONCLUSION: Although sensitivity is lowered by the use of paraffin-embedded specimens, our data indicate that RT-PCR analysis of serial sections from archival SLNs may be helpful in improving detection of occult micrometastases, thus improving staging of patients with melanoma.

Biomarkers, Tumor↗