Screening and Identification of Functional Gene Sets in Aged Kidney-Yang Deficiency Syndrome:cDNA microarray assay
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Objective:To screen and identify functional gene sets in aged kidney-Yang deficiency syndrome by cDNA microarray assay.Methods:Two aged kidney-Yang deficiency patients and two healthy peoples were selected as samples during the epidemiologic survey of the kidney-Yang deficiency syndrome.The differentially expressed genes were screened by applying HG-u133Plus2.0 of Affymetrix GeneChip.The total RNAs were isolated from leukocyte of 4 samples by TR Izolmethod and then purified,reversely transcribed to cDNA with incorporating biotin labeling probe and hybridizated with GeneChip.Picture signals of fluorescence in gene array were scanned and compared differential expression of genes by computer analysis.Results:680 and 503 differentially expressed genes were obtained.The immune response-associated gene sets were found with significance in GO annotated biological processes.Conclusion:Immune factor play an important role in occurrence and development of the kidney-Yang deficiency.Keywords:
Gene chip analysis
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Objective To screen the human pancreatic cancer-associated genes by cDNA microarray.Methods The PCR products of 14 000 genes were spotted onto a chemical-material-coated-glass plates in array.DNAs were fixed onto the glass plate after series of treatments.The total RNAs were isolated from 4 specimens of the pancreatic cancer and the normal tumor-surrounding pancreatic tissues.Both equal quantity RNAs were reversely transcribed to cDNAs and labeled with the fluorescent Cy5-dCTP and Cy3-dCTP to prepare the hybridization probes.The mixed probes were hybridized to the cDNA microarray.After high-stringent washing,the fluorescent signals were scanned by ScanArray 4000 scanner.The values of Cy5-dCTP and Cy3-dCTP on each spot were analyzed and calculated by GenePix Pro 3.0 software.Results By applying this cDNA microarray,189 differentially expressed genes in pancreatic cancer,whose ratios of Cy5/Cy3 were higher than 2.0 or lower than 0.5,were screened out among the 14 000 target genes,comprising 101 known genes and 88 novel gene.Among the known genes,upregulated and downregulated genes were 50 and 51 respectively,including oncogenes and tumor suppression genes,cell cycle related genes,cell skeleton and cinetc-protein related genes,cell apoptosis related genes,DNA synthesis related genes,DNA damage and repair-related genes,transcription factors,receptor related genes,immune related genes,signal transduction related genes,protein translation and synthesis related genes,metabolism related genes.Conclusion The analysis of gene expression profile of tumor based on cDNA microarray can realize high-throughput screening of the genes associated with the pancreatic cancer,and it can help to rapidly explore the genes function.Further analysis of the obtained genes will help to understand the molecular mechanism of the pancreatic cancer.
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Alagille Syndrome
Identification
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Objective To study function and screen differentially expressed genes in development of human glioblastoma by using cDNA microarray.Methods BioStarH140S microarray (including 8 347 old genes and 5 592 novel genes) was adopted and hybridized with probes which were prepared for total RNA. These probes were isolated from 2 cases of normal adult brain tissues and 18 cases of glioma tissues. Differentially expressed genes between normal tissues and glioma tissues were analyzed after scanning microarray with ScanArray4 000. These genes were studied with Hierarchical method and Northern hybridization was used for the function of genes. Results Among the 13 939 target genes, there were multiple differentially expressed genes and 5 genes were significantly up-regulated in glioblastoma. Two of them were testified by Northern-blot.Conclusion cDNA microarray technology is a powerful technique in screening differentially expressed genes; 5 up-regulated genes would be helpful to understand the molecular mechanism of glioblastoma. These genes were tightly related to invasion of glioblastoma and might become molecular index of prognosis of glioblastoma.
Gene chip analysis
Northern blot
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To detect the differentially expressed genes in human polycystic kidney by cDNA microarray.The PCR products of 8398 genes were spotted onto a chip in array. Both mRNAs isolated from polycystic kidney tissue and normal kidney tissue were reversely transcribed to cDNAs with the incorporation of fluorescent dUTP (Cy5-dUTP and Cy3-dUTP) for preparing the hybridization probes. The mixed probes were hybridized to the cDNA microarray. Then the cDNA microarray was scanned for the fluorescent signals and the display of differences between the 2 tissues. IGF1 mRNA, one of the up regulated genes was detected by in situ hybridization technique in the two tissues to validate the result from cDNA microarray.The result indicated that the expressions of 263 genes were up regulated while the expressions of 94 genes were down regulated in the polycystic kidney tissue among the 8398 target genes. Bioinformatical analysis of those genes had been performed. The up-regulated genes were mainly the ones of oncogene, cellular skeleton and movement, apoptosis related protein, cell signal transduction protein, and cytokine. The down regulated genes were mainly the ones of anti-oncogene, DNA binding and transcription factors, cell signal transduction protein, and metabolism protein. The IGF1 mRNA expression detected by in situ hybridization was consequently consistent with the cDNA microarray.cDNA microarray is an effective and quick method for studying differential expressed genes. Three hundred and fifty-seven differentially expressed genes with different functions were revealed in the polycystic kidney tissue, which may play some roles in the progression of polycystic kidney.
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To explore the differences in gene expression between esophageal cancer and cancer around tissue using cDNA microarray and screen new esophageal cancer associated genes.Differentially expressed genes were detected in cancer and cancer around tissue in3cases with esophageal cancer by Northern blotting.The sequences were measured by chain termination method.The result of sequence was homologous checked in GenBank(NLM in USA).In the samples from3cases,there were31differentially expressed genes,among them10up-regulated and21down-regulated.Compared with the data in GenBank2genes were not homologous.These sequences were listed.[Conclusion]With the characteristics of high flux,special and rapid,cDNA differentially expressed microarray is useful for searching the new gene.There are2new differentially expressed genes in esophageal cancer and cancer around tissue.
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Objective:To investigate the differentially expressed genes in hepatic cellular carcinoma(HCC)using cDNA microarray and screen for HCC-related genes.Methods:Total RNA was extracted from normal human liver tissues and hepatic cellular carcinoma tissues.The mRNA was used to prepare probes. The mixed probes were hybridized to the cDNA microarray. After washing, the DNA chips were scanned by laser scanner, The acquired was analyzed.Results:Among the 9182 target genes, 102 genes were identified in HCC tissues that were either over or under-expressed as compared to controls.Forty two genes were up-regulated and 60 down-regulated.Twelve new genes were found in the study. Conclusion: The cDNA microarray for analysis of gene expression profile is a powerful method for screening hepatic cellular carcinoma-related genes.
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A strategy to identify differentially expressed genes during rat liver regeneration using cDNA array
Objective To screen the differentially expressed genes in liver regeneration with cDNA array and to investigate the pathogenesis of acute hepatic failure.Methods Acute liver failure of Sprague-Dawley(SD)rat was induced by 95% hepatectomy.The differentially expressed genes in regenerating rat liver were screened by using a cDNA array representing 1 176 cDNA clusters.Some genes were randomly selected from a pool of differentially expressed genes and subjected to further confirm the result of microarray hybridization by RT-PCR.Results One hundred and thirty-eight genes up-regulated including those of growth factors,PCNA,ribosomal proteins,IL-6 and cdks,which were associated with cell cycle regulation,stress,metabolism and proliferation.Fifty genes down-regulated including metabolic enzyme genes,ER genes,surface antigen genes and so on.Conclusion cDNA array is a powerful tool to explore the gene expression of acute liver failure and provides potential targets for the diagnosis and treatment.
Liver Regeneration
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Objective To study the gene differential expression of taxol resistance cell line OC3/Tax300 of ovarian carcinnoma compared with other parent cell OC3,screen drug-resistance related gene by cDNA microarray, and discuss the reiationship between gene expression difference and drug-resistance.Methods The cDNA retro-transcribed from equal quantity mRNA derived from OC3/Tax300 and OC3 which were labeled with Cy5 and Cy3 fluorescence as probe respectively. The mixed probe were hybridized with BiostarH140S gene expression chip. The acquired image was analyzed by software.Results 134 signitificantly differently expressed genes were screened out, of which up-and down regulated genes were 17 and 117 respectively. They were related to 14 kinds of genes. The down-regulated genes were EBNA-3,COP9, StIP1,and so on. The up-regulated genes were JAK2,HSPS,NADH, and so on.Conclusion These genes related to the mechnisms of drug-resistance of ovarian cancer.
Differential display
Gene chip analysis
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Gene chip analysis
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To investigate the differentially expressed genes in hepatic fibrosis tissues using cDNA microarray and screen for fibrosis-related genes.Total RNA was extracted from normal human liver tissues and liver cirrhosis tissues.The mRNA was used to prepare probes.The mixed probes were hybridized to the cDNA microarray.After washing,the DNA chips were scanned by laster scanner.The acquired image was analyzed.Among the 10.000 target genes,99 genes were identified in liver cirrhosis tissues that were either over or under-expressed as compared to controls.45 genes were up-regulated and 54 down-regulated.9 new genes were found in the study.The results showed that the cDNA microarray for analysis of gene expression profile is a powerful method for screening hepatic fibrosis-related genes.
Hepatic fibrosis
Gene chip analysis
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