In this paper, we developed a rapid and accurate method for the detection of Vibrio parahaemolyticus strains, using multiplex PCR and DNA--DNA hybridization. Multiplex PCR was used to simultaneously amplify three diag...In this paper, we developed a rapid and accurate method for the detection of Vibrio parahaemolyticus strains, using multiplex PCR and DNA--DNA hybridization. Multiplex PCR was used to simultaneously amplify three diagnostic genes （tlh, tdh andfla） that serve as molecular markers of V. parahaemolyticus. Biotinylated PCR products were hybridized to primers immobilized on a microarray, and detected by chemiluminesce with avidin-conjugated alkaline phosphatase. With this method, forty-five samples were tested. Eight known virulent strains （tlh＋/tdh＋/fla＋） and four known avirulent strains （tlh＋/tdh /fla＋） of the V. parahaemolyticus were successfully detected, and no non-specific hybridization and cross-hybridization reaction were found from fifteen closely-related strains （tlh-/tdh-/fla＋） of the Vibrio spp. In addition, all the other eighteen strains of non-Vibrio bacteria （tlh-/tdh /fla-） gave negative results. The DNA microarray successfully distinguished V. parahaemolyticus from other Vibrio spp. The results demonstrated that this was an efficient and robust method for identifying virulent strains of V. parahaemolyticus.展开更多
Objective： The aim of this study was to investigate the MRP-1/CD9mRNA expression in lung cancer and normal lung tissues and the relationship between its expression and pathologic grades, clinical stages, metastasis a...Objective： The aim of this study was to investigate the MRP-1/CD9mRNA expression in lung cancer and normal lung tissues and the relationship between its expression and pathologic grades, clinical stages, metastasis and prognosis. Methods： To observe MRP-1/C9mRNA expression, tissue microarray （TMA） containing 54 lung cancers and 10 normal lung tissues was prepared and Fluorescence in situ hybridization was used. Results： The positive rate of MRP-1/CD9 expression was 48.1% in lung cancer, lower than that of normal lung tissues. The statistical difference was significant （P〈0.05）. Its protein expression had no relationship with the patients＇ ages, sex and the macroscopic type of tumor, but had relationships with the histological type, clinical stage, differentiated degree and metastasis. The expression in non-small cell lung cancer （NSCLC） was higher than that in small cell lung cancer （SCLC）; in well-moderately differentiated group was higher than that in poorly differentiated group; Earlier period group （I＋II） was higher than in later period group （Ⅲ＋Ⅳ）; and in group without lymphoid metastasis was higher than in patients with lymphoid metastasis. Conclusion： The progression of the lung cancer maybe related with the descended MRP-1/Cd9 expression, which may be useful in evaluating the prognosis of cancer patients.展开更多
Acute lymphoblastic leukemia (ALL) is the most common malignancy in children, with the majority of cases being of precursor B-cell phenoltype. Conventional cytogenetic analysis plays an important role in the diagnosis...Acute lymphoblastic leukemia (ALL) is the most common malignancy in children, with the majority of cases being of precursor B-cell phenoltype. Conventional cytogenetic analysis plays an important role in the diagnosis of B-cell ALL, identifying characteristic chromosomal abnormalities associated with a given prognosis therein facilitating optimized treatment. The more recent introduction of microarray technology to the analysis of B-cell ALL has afforded both higher resolution for the detection of known abnormalities and an ability to identify novel copy number abnormalities (CNAs) with potential clinical relevance. In the current study, microarray analysis was performed on 20 cytogenetically abnormal B-cell ALL cases (10 pediatric and 10 adult), while a novel microarray-based balanced-translocation detection methodology (translocation CGH or tCGH) was applied to that subset of cases with a known or suspected recurrent balanced translocation. Standard microarray analysis identified that CNAs was not detected by previous conventional cytogenetics in 75% (15/20) cases. tCGH identified 9/9 (100%) balanced translocations defining BCR/ABL1 (x4), ETV6/RUNX1 (x3), and MLL/AFF1 (x2) breakpoints with high resolution. The results illustrate the improved molecular detail afforded by these technologies and a comparison of translocation breakpoints, CNAs and patient age offers new insights into tumor biology with potential prognostic significance.展开更多
Objective To investigate the expression of Survivin mRNA in lung cancer tissue microarray （TMA） by fluorescence in .situ hybridization （FISH） method, and determine the role and significance of it in lung cancer ge...Objective To investigate the expression of Survivin mRNA in lung cancer tissue microarray （TMA） by fluorescence in .situ hybridization （FISH） method, and determine the role and significance of it in lung cancer genesis and progress. Methods The expression of Survivin mRNA was detected by FISH method and TMA technology. Fifty-four cases of lung cancer and 10 cases of normal lung tissue were examined. Survivin mRNA was expressed in 66.7% （36/54） of lung cancer; the positive ratio of lung cancer was significantly higher than that of normal lung tissue （0/10;X^2= 15.238, P 〈 0.05）. The positive ratio of Survivin mRNA was significantly higher in poor differentiated cancer （20/24, 83.3% ） than moderate and well differentiated cancer （16/30, 53.3%; X^2 = 5.40, P 〈 0.05）. The positive ratio of Survivin mRNA was significantly higher in group with lymph node metastasis （27/32, 84.4%） than without lymph node metastasis （9/22, 40.9%; X^2= 11.084, P 〈 0.05）. The positive ratio of Survivin mRNA was significantly higher in stage Ⅲ-Ⅳ（12/13, 92.3%） than stage Ⅰ- Ⅱ （24/41,58.5%; X^2=5.066, P〈 0.05）. Conclusion Survivin mRNA highly expresses in lung cancer, which is related to the progress and malignant behavior. Survivin may play a promoting role in lung cancer genesis and progress and provide a basis for estimating prognosis and treatment.展开更多
Microduplications are normally invisible under microscopy and were not recognized before chromosomal microarray testing was available. Although it is difficult to confirm the orientation of duplicated segments by stan...Microduplications are normally invisible under microscopy and were not recognized before chromosomal microarray testing was available. Although it is difficult to confirm the orientation of duplicated segments by standard fluorescence in situ hybridization(FISH), our data indicates that fiber-FISH analysis has the potential to reveal the orientation of duplicated and triplicated segments of chromosomes. Recurrent microduplications reciprocal to microdeletions show tandem orientations of the duplicated segments, which is consistent with a non-allelic homologous recombination mechanism. Several random duplications showed tandem configurations and inverted duplications are rare. Further analysis is required to fully elucidate the basic mechanisms underlying such duplications/triplications.展开更多
Objective: To evaluate p63 expression at mRNA transcripts and protein levels in lung squamous cell cancer (SCC), adenocarcinoma, large cell lung cancer (LCLC) and small cell lung cancer (SCLC) and their matched metast...Objective: To evaluate p63 expression at mRNA transcripts and protein levels in lung squamous cell cancer (SCC), adenocarcinoma, large cell lung cancer (LCLC) and small cell lung cancer (SCLC) and their matched metastatic tumors. The association between p63 expression and p63 locus at chromosomal 3q27 q29 was also investigated. Methods: p63 mRNA expression levels in a large series of lung cancers including SCC, adenocarcinoma, LCLC, SCLC and their matched metastatic tumors were analyzed by cDNA microarray technology. A tissue microarray from 150 primary lung cancer specimens was constructed and used for immunohistochemical detection of p63 protein expression. Chromosomal imbalances at the p63 locus in 70 primary lung cancers samples were studied by comparative genomic hybridization (CGH) technology. Results: mRNA levels were 10 fold in SCC compared to LCLC, SCLC, and adenocarcinoma. Interestingly, the mRNA expression of p63 in metastatic carcinomas was significantly higher than that in their matched primary tumors ( P <0 001). Immunohistochemistry demonstrated that p63 expression was 94.64% in SCC but only 1 79% in lung adenocarcinoma and 2 of 4 LCLC were positive staining. All the results in of SCLC were negative. There was a statistically significant difference for p63 positivity between pT1 tumors and those of higher stage ( P =0 035). The CGH results indicated that p63 locus at chromosomal 3q27 q29 was overrepresented in SCC. p63 immunopositivity correlated significantly with pronounced gains of the p63 locus at chromosomal 3q27 q29 (P=0.0001), indicating that strong expression of p63 in lung SCC correlated with increased gene amplification. Conclusion: p63 might play an important role not only in squamous differentiation of lung cancer but also in tumor development and progression.展开更多
Objective: To identify the differential gene expression profiles between the normal and aspermia human testes utilizing cDNA microarray. Methods: cDNA probes were prepared by labeling mRNA of aspermia testes tissues w...Objective: To identify the differential gene expression profiles between the normal and aspermia human testes utilizing cDNA microarray. Methods: cDNA probes were prepared by labeling mRNA of aspermia testes tissues with Cy5-dUTP and mRNA of normal testes tissues with Cy3-dUTP respectively through reverse transcription. The mixed cDNA probes were then hybridized with 4096 cDNA arrays (4096 unique human cDNA sequences), and the fluorescent signals were scanned by ScanArray 3000 scanner (General Scanning, Inc.). The values of Cy5-dUTP and Cy3-dUTP on each spot were analyzed and calculated by ImaGene 3.0 software (BioDiscovery, Inc.). Differentially expressed genes were screened according to the criterion that the absolute value of natural logarithm of the ratio of Cy5-dUTP to Cy3-dUTP was greater-than 2.0 or less-than 0.5. A randomly chosen gene RAP1A was studied by in situ hybridization to evaluate the accuracy of the results. Results: 623 differential expressed genes related to aspermia were found. There were 303 up-expressed genes and 320 down-expressed genes. A distinct up-expressed gene RAP1A was confirmed by in situ hybridization. Conclusions: Screening the differential gene expression profiles between the normal and aspermia human testis by cDNA microarray can be used in the study of aspermia-related genes and the further research due to its properties, RAP1A may play some roles in the development and progression of aspermia.展开更多
A series of human tissue samples and cultured cell lines were formalin-fixed and paraffin-embedded. Specimen cylinder （1.2 -1.8ram） were punched by a modified bone marrow biopsy needle and arrayed on a recipient par...A series of human tissue samples and cultured cell lines were formalin-fixed and paraffin-embedded. Specimen cylinder （1.2 -1.8ram） were punched by a modified bone marrow biopsy needle and arrayed on a recipient paraffin block. Microscopic analysis on the sections from this tissue microarray （ TMA ） block demonstrated that the spots of tissues and cells were well preserved, and the cultured cell samples were successfully embedded from 5 × 104 to 2 × 105 in number. These TMA sections were also suitable for immunohistochemistry and RNA in situ hybridization.展开更多
目的探讨染色体微阵列分析(CMA)、荧光原位杂交(FISH)和染色体核型分析技术在疑难病例中的诊断价值。方法以新生儿科的一名住院患者为研究对象,采集外周血进行染色体核型分析,用CMA技术检测基因组拷贝数变异(copy number variations,CNV...目的探讨染色体微阵列分析(CMA)、荧光原位杂交(FISH)和染色体核型分析技术在疑难病例中的诊断价值。方法以新生儿科的一名住院患者为研究对象,采集外周血进行染色体核型分析,用CMA技术检测基因组拷贝数变异(copy number variations,CNVs),并用FISH技术对存在的微缺失/微重复位点进行验证;因CMA检测结果提示患者存在基因组的微缺失/微重复,故采集患者父母的外周血进行染色体核型分析,以辅助诊断患者染色体异常的遗传来源。结果患者染色体核型分析结果为46,XY,CMA检测结果提示患者15号染色体15q11.2q12区段存在3.8 Mb缺失,21号染色体21q11.2q22.11区段存在19.8 Mb重复,患者染色体核型分析结果存在漏诊。患者母亲染色体核型分析结果为46,XX,患者父亲染色体核型分析结果为46,XY,t(15;21)(q12;q22.11)。通过FISH技术验证确认患者染色体核型为非平衡易位,正确结果应为:46,XY,-15,+der(21)t(15;21).ish der(21)t(15;21)(q12;q22.11)pat。结论对染色体核型分析可疑的核型结果,应联合多种分子技术进行检测,降低单独应用染色体核型分析的漏诊风险。展开更多
基金financial supports from National High Technology Research and Development Program of China(No.2007AA10Z430)National Natural Science Foundation of China(No.30700535)Program for New Century Excellent Talents in Fujian Province University,and Fok Ying Tong Education Foundation(No.111032)
文摘In this paper, we developed a rapid and accurate method for the detection of Vibrio parahaemolyticus strains, using multiplex PCR and DNA--DNA hybridization. Multiplex PCR was used to simultaneously amplify three diagnostic genes （tlh, tdh andfla） that serve as molecular markers of V. parahaemolyticus. Biotinylated PCR products were hybridized to primers immobilized on a microarray, and detected by chemiluminesce with avidin-conjugated alkaline phosphatase. With this method, forty-five samples were tested. Eight known virulent strains （tlh＋/tdh＋/fla＋） and four known avirulent strains （tlh＋/tdh /fla＋） of the V. parahaemolyticus were successfully detected, and no non-specific hybridization and cross-hybridization reaction were found from fifteen closely-related strains （tlh-/tdh-/fla＋） of the Vibrio spp. In addition, all the other eighteen strains of non-Vibrio bacteria （tlh-/tdh /fla-） gave negative results. The DNA microarray successfully distinguished V. parahaemolyticus from other Vibrio spp. The results demonstrated that this was an efficient and robust method for identifying virulent strains of V. parahaemolyticus.
基金This work was supported by a grant from Tianjin Science and Technology Committee (No. 033804211)
文摘Objective： The aim of this study was to investigate the MRP-1/CD9mRNA expression in lung cancer and normal lung tissues and the relationship between its expression and pathologic grades, clinical stages, metastasis and prognosis. Methods： To observe MRP-1/C9mRNA expression, tissue microarray （TMA） containing 54 lung cancers and 10 normal lung tissues was prepared and Fluorescence in situ hybridization was used. Results： The positive rate of MRP-1/CD9 expression was 48.1% in lung cancer, lower than that of normal lung tissues. The statistical difference was significant （P〈0.05）. Its protein expression had no relationship with the patients＇ ages, sex and the macroscopic type of tumor, but had relationships with the histological type, clinical stage, differentiated degree and metastasis. The expression in non-small cell lung cancer （NSCLC） was higher than that in small cell lung cancer （SCLC）; in well-moderately differentiated group was higher than that in poorly differentiated group; Earlier period group （I＋II） was higher than in later period group （Ⅲ＋Ⅳ）; and in group without lymphoid metastasis was higher than in patients with lymphoid metastasis. Conclusion： The progression of the lung cancer maybe related with the descended MRP-1/Cd9 expression, which may be useful in evaluating the prognosis of cancer patients.
文摘Acute lymphoblastic leukemia (ALL) is the most common malignancy in children, with the majority of cases being of precursor B-cell phenoltype. Conventional cytogenetic analysis plays an important role in the diagnosis of B-cell ALL, identifying characteristic chromosomal abnormalities associated with a given prognosis therein facilitating optimized treatment. The more recent introduction of microarray technology to the analysis of B-cell ALL has afforded both higher resolution for the detection of known abnormalities and an ability to identify novel copy number abnormalities (CNAs) with potential clinical relevance. In the current study, microarray analysis was performed on 20 cytogenetically abnormal B-cell ALL cases (10 pediatric and 10 adult), while a novel microarray-based balanced-translocation detection methodology (translocation CGH or tCGH) was applied to that subset of cases with a known or suspected recurrent balanced translocation. Standard microarray analysis identified that CNAs was not detected by previous conventional cytogenetics in 75% (15/20) cases. tCGH identified 9/9 (100%) balanced translocations defining BCR/ABL1 (x4), ETV6/RUNX1 (x3), and MLL/AFF1 (x2) breakpoints with high resolution. The results illustrate the improved molecular detail afforded by these technologies and a comparison of translocation breakpoints, CNAs and patient age offers new insights into tumor biology with potential prognostic significance.
基金Supported byaK eyProjectofTianjin ScientificCom m ittee(033804211).
文摘Objective To investigate the expression of Survivin mRNA in lung cancer tissue microarray （TMA） by fluorescence in .situ hybridization （FISH） method, and determine the role and significance of it in lung cancer genesis and progress. Methods The expression of Survivin mRNA was detected by FISH method and TMA technology. Fifty-four cases of lung cancer and 10 cases of normal lung tissue were examined. Survivin mRNA was expressed in 66.7% （36/54） of lung cancer; the positive ratio of lung cancer was significantly higher than that of normal lung tissue （0/10;X^2= 15.238, P 〈 0.05）. The positive ratio of Survivin mRNA was significantly higher in poor differentiated cancer （20/24, 83.3% ） than moderate and well differentiated cancer （16/30, 53.3%; X^2 = 5.40, P 〈 0.05）. The positive ratio of Survivin mRNA was significantly higher in group with lymph node metastasis （27/32, 84.4%） than without lymph node metastasis （9/22, 40.9%; X^2= 11.084, P 〈 0.05）. The positive ratio of Survivin mRNA was significantly higher in stage Ⅲ-Ⅳ（12/13, 92.3%） than stage Ⅰ- Ⅱ （24/41,58.5%; X^2=5.066, P〈 0.05）. Conclusion Survivin mRNA highly expresses in lung cancer, which is related to the progress and malignant behavior. Survivin may play a promoting role in lung cancer genesis and progress and provide a basis for estimating prognosis and treatment.
文摘Microduplications are normally invisible under microscopy and were not recognized before chromosomal microarray testing was available. Although it is difficult to confirm the orientation of duplicated segments by standard fluorescence in situ hybridization(FISH), our data indicates that fiber-FISH analysis has the potential to reveal the orientation of duplicated and triplicated segments of chromosomes. Recurrent microduplications reciprocal to microdeletions show tandem orientations of the duplicated segments, which is consistent with a non-allelic homologous recombination mechanism. Several random duplications showed tandem configurations and inverted duplications are rare. Further analysis is required to fully elucidate the basic mechanisms underlying such duplications/triplications.
文摘Objective: To evaluate p63 expression at mRNA transcripts and protein levels in lung squamous cell cancer (SCC), adenocarcinoma, large cell lung cancer (LCLC) and small cell lung cancer (SCLC) and their matched metastatic tumors. The association between p63 expression and p63 locus at chromosomal 3q27 q29 was also investigated. Methods: p63 mRNA expression levels in a large series of lung cancers including SCC, adenocarcinoma, LCLC, SCLC and their matched metastatic tumors were analyzed by cDNA microarray technology. A tissue microarray from 150 primary lung cancer specimens was constructed and used for immunohistochemical detection of p63 protein expression. Chromosomal imbalances at the p63 locus in 70 primary lung cancers samples were studied by comparative genomic hybridization (CGH) technology. Results: mRNA levels were 10 fold in SCC compared to LCLC, SCLC, and adenocarcinoma. Interestingly, the mRNA expression of p63 in metastatic carcinomas was significantly higher than that in their matched primary tumors ( P <0 001). Immunohistochemistry demonstrated that p63 expression was 94.64% in SCC but only 1 79% in lung adenocarcinoma and 2 of 4 LCLC were positive staining. All the results in of SCLC were negative. There was a statistically significant difference for p63 positivity between pT1 tumors and those of higher stage ( P =0 035). The CGH results indicated that p63 locus at chromosomal 3q27 q29 was overrepresented in SCC. p63 immunopositivity correlated significantly with pronounced gains of the p63 locus at chromosomal 3q27 q29 (P=0.0001), indicating that strong expression of p63 in lung SCC correlated with increased gene amplification. Conclusion: p63 might play an important role not only in squamous differentiation of lung cancer but also in tumor development and progression.
文摘Objective: To identify the differential gene expression profiles between the normal and aspermia human testes utilizing cDNA microarray. Methods: cDNA probes were prepared by labeling mRNA of aspermia testes tissues with Cy5-dUTP and mRNA of normal testes tissues with Cy3-dUTP respectively through reverse transcription. The mixed cDNA probes were then hybridized with 4096 cDNA arrays (4096 unique human cDNA sequences), and the fluorescent signals were scanned by ScanArray 3000 scanner (General Scanning, Inc.). The values of Cy5-dUTP and Cy3-dUTP on each spot were analyzed and calculated by ImaGene 3.0 software (BioDiscovery, Inc.). Differentially expressed genes were screened according to the criterion that the absolute value of natural logarithm of the ratio of Cy5-dUTP to Cy3-dUTP was greater-than 2.0 or less-than 0.5. A randomly chosen gene RAP1A was studied by in situ hybridization to evaluate the accuracy of the results. Results: 623 differential expressed genes related to aspermia were found. There were 303 up-expressed genes and 320 down-expressed genes. A distinct up-expressed gene RAP1A was confirmed by in situ hybridization. Conclusions: Screening the differential gene expression profiles between the normal and aspermia human testis by cDNA microarray can be used in the study of aspermia-related genes and the further research due to its properties, RAP1A may play some roles in the development and progression of aspermia.
文摘A series of human tissue samples and cultured cell lines were formalin-fixed and paraffin-embedded. Specimen cylinder （1.2 -1.8ram） were punched by a modified bone marrow biopsy needle and arrayed on a recipient paraffin block. Microscopic analysis on the sections from this tissue microarray （ TMA ） block demonstrated that the spots of tissues and cells were well preserved, and the cultured cell samples were successfully embedded from 5 × 104 to 2 × 105 in number. These TMA sections were also suitable for immunohistochemistry and RNA in situ hybridization.
