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1.
Springerplus ; 4: 7, 2015.
Article in English | MEDLINE | ID: mdl-25674493

ABSTRACT

We compared the performance of the 3D-Gene® mutation assay (3D-Gene® KRAS mutation assay kit) with the Scorpion-ARMS (therascreen® KRAS RGQ PCR Kit) and Luminex (MEBGEN™ KRAS kit) assays for the detection of KRAS mutations in formalin-fixed, paraffin-embedded tissue samples from 150 patients diagnosed with colorectal cancer. DNA was extracted from the paraffin-embedded tissue samples with or without macrodissection under hematoxylin and eosin staining and the KRAS mutation status was independently determined using these assays. Discordant results were re-analyzed by Sanger sequencing. Mutation detection analysis was successfully performed in all 150 specimens using the 3D-Gene® mutation assay without an invalid case. The concordance rate between the 3D-Gene® mutation assay and Scorpion-ARMS or Luminex was 98.7% (148/150). KRAS mutations were detected at a frequency of 35.3% (53/150) in colorectal cancer specimens. Three discrepant cases were found between the three assays. Overall, our results demonstrate a high concordance rate of between the 3D-Gene® mutation assay and the two existing in-vitro diagnostics kits. All three assays proved to be validated methods for detecting clinically significant KRAS mutations in paraffin-embedded tissue samples.

2.
PLoS One ; 10(2): e0118220, 2015.
Article in English | MEDLINE | ID: mdl-25706130

ABSTRACT

It is difficult to detect pancreatic cancer or biliary-tract cancer at an early stage using current diagnostic technology. Utilizing microRNA (miRNA) markers that are stably present in peripheral blood, we aimed to identify pancreatic and biliary-tract cancers in patients. With "3D-Gene", a highly sensitive microarray, we examined comprehensive miRNA expression profiles in 571 serum samples obtained from healthy patients, patients with pancreatic, biliary-tract, or other digestive cancers, and patients with non-malignant abnormalities in the pancreas or biliary tract. The samples were randomly divided into training and test cohorts, and candidate miRNA markers were independently evaluated. We found 81 miRNAs for pancreatic cancer and 66 miRNAs for biliary-tract cancer that showed statistically different expression compared with healthy controls. Among those markers, 55 miRNAs were common in both the pancreatic and biliary-tract cancer samples. The previously reported miR-125a-3p was one of the common markers; however, it was also expressed in other types of digestive-tract cancers, suggesting that it is not specific to cancer types. In order to discriminate the pancreato-biliary cancers from all other clinical conditions including the healthy controls, non-malignant abnormalities, and other types of cancers, we developed a diagnostic index using expression profiles of the 10 most significant miRNAs. A combination of eight miRNAs (miR-6075, miR-4294, miR-6880-5p, miR-6799-5p, miR-125a-3p, miR-4530, miR-6836-3p, and miR-4476) achieved a sensitivity, specificity, accuracy and AUC of 80.3%, 97.6%, 91.6% and 0.953, respectively. In contrast, CA19-9 and CEA gave sensitivities of 65.6% and 40.0%, specificities of 92.9% and 88.6%, and accuracies of 82.1% and 71.8%, respectively, in the same test cohort. This diagnostic index identified 18/21 operable pancreatic cancers and 38/48 operable biliary-tract cancers in the entire cohort. Our results suggest that the assessment of these miRNA markers is clinically valuable to identify patients with pancreato-biliary cancers who could benefit from surgical intervention.


Subject(s)
Biliary Tract Neoplasms/genetics , Biomarkers, Tumor/genetics , MicroRNAs/genetics , Pancreatic Neoplasms/genetics , Aged , Biliary Tract Neoplasms/blood , Biliary Tract Neoplasms/diagnosis , Biomarkers, Tumor/blood , Diagnosis, Differential , Female , Gene Expression Profiling/methods , Gene Expression Regulation, Neoplastic , Humans , Male , MicroRNAs/blood , Middle Aged , Oligonucleotide Array Sequence Analysis , Pancreatic Neoplasms/blood , Pancreatic Neoplasms/diagnosis , Reproducibility of Results , Sensitivity and Specificity
3.
Anal Biochem ; 472: 75-83, 2015 Mar 01.
Article in English | MEDLINE | ID: mdl-25481737

ABSTRACT

RNA external standards, although important to ensure equivalence across many microarray platforms, have yet to be fully implemented in the research community. In this article, a set of unique RNA external standards (or RNA standards) and probe pairs that were added to total RNA in the samples before amplification and labeling are described. Concentration-response curves of RNA external standards were used across multiple commercial DNA microarray platforms and/or quantitative real-time polymerase chain reaction (RT-PCR) and next-generation sequencing to identify problematic assays and potential sources of variation in the analytical process. A variety of standards can be added in a range of concentrations spanning high and low abundances, thereby enabling the evaluation of assay performance across the expected range of concentrations found in a clinical sample. Using this approach, we show that we are able to confirm the dynamic range and the limit of detection for each DNA microarray platform, RT-PCR protocol, and next-generation sequencer. In addition, the combination of a series of standards and their probes was investigated on each platform, demonstrating that multiplatform calibration and validation is possible.


Subject(s)
Models, Chemical , Oligonucleotide Array Sequence Analysis/methods , Oligonucleotide Array Sequence Analysis/standards , RNA Probes/chemistry , Reference Standards
4.
J Appl Toxicol ; 31(7): 671-7, 2011 Oct.
Article in English | MEDLINE | ID: mdl-21328587

ABSTRACT

BIRB-796, a selective inhibitor of p38 mitogen-activated protein kinase, has entered clinical trials for the treatment of autoimmune diseases. Levels of alanine transaminase, a biomarker of hepatic toxicity in clinical pathology, were found to be increased in Crohn's disease patients treated with BIRB-796. The purpose of the present study was to clarify the molecular mechanism(s) of this hepatotoxicity. A toxicogenomic analysis using a highly sensitive DNA chip, 3D-Gene™ Mouse Oligo chip 24k, indicated that BIRB-796 treatment activated the nuclear factor (erythroid-derived 2)-like 2 signaling pathway, which plays a key role in the response to oxidative stress. A reactive intermediate of BIRB-796 was detected by the glutathione-trapping method using mouse and human liver microsomes. The production of this reactive metabolite in the liver may be one of the causes of BIRB-796's hepatotoxicity.


Subject(s)
Chemical and Drug Induced Liver Injury/pathology , Naphthalenes/toxicity , Pyrazoles/toxicity , p38 Mitogen-Activated Protein Kinases/metabolism , Animals , Chromatography, Liquid , Down-Regulation , Glutathione/metabolism , Humans , Male , Mice , Mice, Inbred ICR , Microsomes, Liver/drug effects , Microsomes, Liver/metabolism , NF-E2-Related Factor 2/genetics , NF-E2-Related Factor 2/metabolism , Oligonucleotide Array Sequence Analysis/methods , Oxidative Stress/drug effects , RNA, Messenger , Signal Transduction , Tandem Mass Spectrometry , Toxicogenetics/methods , Up-Regulation , p38 Mitogen-Activated Protein Kinases/antagonists & inhibitors , p38 Mitogen-Activated Protein Kinases/genetics
5.
Drug Metab Pharmacokinet ; 26(3): 228-35, 2011 Jun.
Article in English | MEDLINE | ID: mdl-21273731

ABSTRACT

Cytochromes P450 (P450s or CYPs) are a gene family of highly homologous genes and include the CYP1-4 family, which is relevant to drug metabolism. In the cynomolgus monkey (which is frequently used in drug metabolism studies), numerous CYPs (mfCYPs) have been identified in the CYP1-4 family. DNA microarrays are useful for high-throughput screening assays; however, there is a potential problem with cross-hybridization of highly homologous genes in the gene family. This problem might be solved with the use of low-density DNA microarrays, with which specific validation can be performed for the genes on the microarray. We have developed a DNA microarray for the 20 mfCYPs and have evaluated and validated its specificity and usefulness. First, in both DNA microarray and quantitative polymerase chain reaction (qPCR) analyses, hepatic expression of each mfCYP correlated well, and similar tissue expression patterns were observed for five representative mfCYPs, confirming the specificity of the DNA microarray. Second, the usefulness of this DNA microarray was validated by induction analysis of mfCYPs in primary hepatocytes, which successfully detected known responders, but also novel responders (mfCYP2C43, mfCYP2C75, and mfCYP3A5 for rifampicin), as confirmed by qPCR analysis. This DNA microarray can thus be utilized for high-throughput assays during drug development.


Subject(s)
Cytochrome P-450 Enzyme System/genetics , Drug Evaluation, Preclinical/methods , Enzyme Induction/drug effects , Macaca fascicularis , Oligonucleotide Array Sequence Analysis/methods , Animal Structures/metabolism , Animals , Cells, Cultured , Enzyme Induction/genetics , Gene Expression/drug effects , Gene Expression/genetics , Hepatocytes/drug effects , Hepatocytes/metabolism , Ileum/metabolism , Isoenzymes/genetics , Jejunum/metabolism , Kidney Cortex/metabolism , Kidney Medulla/metabolism , Liver/metabolism , Macaca fascicularis/genetics , Macaca fascicularis/metabolism , Male , Omeprazole/pharmacology , Rifampin/pharmacology
6.
J Cancer Res Clin Oncol ; 134(12): 1363-9, 2008 Dec.
Article in English | MEDLINE | ID: mdl-18483744

ABSTRACT

PURPOSE: Renal cell carcinoma (RCC) is characterized by a variable and unpredictable clinical course. Thus, accurate prediction of the prognosis is important in clinical settings. We conducted microarray-based study to identify a novel prognostic marker in conventional RCC. PATIENTS AND METHODS: The present study included the patients surgically treated at Kyoto University Hospital. Gene expression profiling of 39 samples was carried out to select candidate prognostic markers. Quantitative real-time PCR of 65 samples confirmed the microarray experiment results. Finally, we evaluated the significance of potential markers at their protein expression level by immunohistochemically analyzing 230 conventional RCC patients. RESULTS: Using expression profiling analysis, we identified 14 candidate genes whose expression levels predicted unfavorable disease-specific survival. Next, we examined the expression levels of nine candidate genes by quantitative real-time PCR and selected CUB-domain containing protein 1 (CDCP1) for further immunohistochemical analysis. Positive staining for CDCP1 inversely correlated with disease-specific and recurrence-free survivals. In multivariate analysis including clinical/pathological factors, CDCP1 staining was a significant predictor of disease-specific and recurrence-free survivals. CONCLUSIONS: We identified CDCP1 as a potential prognostic marker for conventional RCC. Further studies might be required to confirm the prognostic value of CDCP1 and to understand its function in RCC progression.


Subject(s)
Adenocarcinoma, Clear Cell/genetics , Antigens, CD/genetics , Biomarkers, Tumor/genetics , Carcinoma, Renal Cell/genetics , Cell Adhesion Molecules/genetics , Gene Expression Profiling , Kidney Neoplasms/genetics , Neoplasm Proteins/genetics , Oligonucleotide Array Sequence Analysis/methods , Adenocarcinoma, Clear Cell/diagnosis , Adenocarcinoma, Clear Cell/metabolism , Adult , Aged , Aged, 80 and over , Antigens, CD/metabolism , Antigens, Neoplasm , Biomarkers, Tumor/metabolism , Carcinoma, Renal Cell/diagnosis , Carcinoma, Renal Cell/metabolism , Cell Adhesion Molecules/metabolism , Female , Humans , Immunoenzyme Techniques , Kidney Neoplasms/diagnosis , Kidney Neoplasms/metabolism , Male , Middle Aged , Neoplasm Proteins/metabolism , Neoplasm Staging , Prognosis , RNA, Messenger/genetics , RNA, Messenger/metabolism , RNA, Neoplasm/genetics , RNA, Neoplasm/metabolism , Reverse Transcriptase Polymerase Chain Reaction , Survival Rate
7.
Clin Cancer Res ; 14(9): 2579-87, 2008 May 01.
Article in English | MEDLINE | ID: mdl-18451219

ABSTRACT

PURPOSE: The purpose of this study was to identify proteins that are potentially involved in the tumor invasion of bladder cancer. EXPERIMENTAL DESIGN: We searched for the candidate proteins by comparing the profiles of secreted proteins among the poorly invasive human bladder carcinoma cell line RT112 and the highly invasive cell line T24. The proteins isolated from cell culture supernatants were identified by shotgun proteomics. We found that CXCL1 is related to the tumor invasion of bladder cancer cells. We also evaluated whether the amount of the chemokine CXCL1 in the urine would be a potential marker for predicting the existence of invasive bladder tumors. RESULTS: Higher amount of CXCL1 was secreted from highly invasive bladder carcinoma cell lines and this chemokine modulated the invasive ability of those cells in vitro. It was revealed that CXCL1 regulated the expression of matrix metalloproteinase-13 in vitro and higher expression of CXCL1 was associated with higher pathologic stages in bladder cancer in vivo. We also showed that urinary CXCL1 levels were significantly higher in patients with invasive bladder cancer (pT1-4) than those with noninvasive pTa tumors (P = 0.0028) and normal control (P < 0.0001). Finally, it was shown that CXCL1 was an independent factor for predicting the bladder cancer with invasive phenotype. CONCLUSIONS: Our results suggest that CXCL1 modulates the invasive abilities of bladder cancer cells and this chemokine may be a potential candidate of urinary biomarker for invasive bladder cancer and a possible therapeutic target for preventing tumor invasion.


Subject(s)
Biomarkers, Tumor/metabolism , Chemokine CXCL1/metabolism , Matrix Metalloproteinase 13/metabolism , Urinary Bladder Neoplasms/metabolism , Biomarkers, Tumor/urine , Cell Line, Tumor , Chemokine CXCL1/urine , Disease Progression , Humans , Neoplasm Invasiveness , Oligonucleotide Array Sequence Analysis , Proteomics , Up-Regulation , Urinary Bladder Neoplasms/pathology , Urinary Bladder Neoplasms/urine
8.
BMC Genomics ; 8: 95, 2007 Apr 05.
Article in English | MEDLINE | ID: mdl-17408496

ABSTRACT

BACKGROUND: Mycotoxins are fungal secondary metabolites commonly present in feed and food, and are widely regarded as hazardous contaminants. Citrinin, one of the very well known mycotoxins that was first isolated from Penicillium citrinum, is produced by more than 10 kinds of fungi, and is possibly spread all over the world. However, the information on the action mechanism of the toxin is limited. Thus, we investigated the citrinin-induced genomic response for evaluating its toxicity. RESULTS: Citrinin inhibited growth of yeast cells at a concentration higher than 100 ppm. We monitored the citrinin-induced mRNA expression profiles in yeast using the ORF DNA microarray and Oligo DNA microarray, and the expression profiles were compared with those of the other stress-inducing agents. Results obtained from both microarray experiments clustered together, but were different from those of the mycotoxin patulin. The oxidative stress response genes--AADs, FLR1, OYE3, GRE2, and MET17--were significantly induced. In the functional category, expression of genes involved in "metabolism", "cell rescue, defense and virulence", and "energy" were significantly activated. In the category of "metabolism", genes involved in the glutathione synthesis pathway were activated, and in the category of "cell rescue, defense and virulence", the ABC transporter genes were induced. To alleviate the induced stress, these cells might pump out the citrinin after modification with glutathione. While, the citrinin treatment did not induce the genes involved in the DNA repair. CONCLUSION: Results from both microarray studies suggest that citrinin treatment induced oxidative stress in yeast cells. The genotoxicity was less severe than the patulin, suggesting that citrinin is less toxic than patulin. The reproducibility of the expression profiles was much better with the Oligo DNA microarray. However, the Oligo DNA microarray did not completely overcome cross hybridization.


Subject(s)
Citrinin/toxicity , Gene Expression Regulation, Fungal/drug effects , Oligonucleotide Array Sequence Analysis/methods , Oligonucleotides , Open Reading Frames , Saccharomyces cerevisiae/drug effects , Toxicity Tests/methods , Cluster Analysis , Genes, Fungal , Glutathione/metabolism , Metabolic Networks and Pathways/drug effects , Metabolic Networks and Pathways/genetics , Methionine/metabolism , Models, Biological , Oligonucleotides/analysis , Patulin/toxicity , Saccharomyces cerevisiae/genetics
9.
Oncology ; 73(5-6): 366-75, 2007.
Article in English | MEDLINE | ID: mdl-18511875

ABSTRACT

OBJECTIVES: Gene expression profiling using pretreatment biopsies has been limited due to their small sample sizes. This study evaluated the usefulness of an ultrasensitive new DNA microarray chip, which has a unique array structure, for the clinical diagnosis of esophageal cancer using preoperative biopsies. METHODS: Paired cancer and normal esophageal epithelial tissues from 56 patients who underwent esophagectomy and from 48 patients who underwent preoperative endoscopy were studied. Among 2 feature gene sets selected by a reference DNA chip discriminating malignant status of samples, 20 feature genes were selected for the development of the new DNA chip. The new DNA chip was hybridized with 0.1 mug of total RNA per slide without RNA amplification. RESULTS: Twenty feature genes, including RRM-2 and XRCC-3, for the new DNA chip could discriminate cancer from noncancer at a 95.2% rate of accuracy in 42 biopsies (sensitivity 95.7%, specificity 94.7%). A receiver operating characteristic (ROC) curve analysis showed that the area under ROC curve for the prediction was 0.966. CONCLUSIONS: The gene expression profiles from the preoperative biopsies could diagnose esophageal cancer accurately, using the ultrasensitive DNA chip without RNA amplification. This new DNA chip technology might contribute further to the development of customized therapeutic strategies for various cancer patients.


Subject(s)
Carcinoma, Squamous Cell/pathology , Esophageal Neoplasms/pathology , Esophageal Neoplasms/surgery , Gene Expression Profiling , Oligonucleotide Array Sequence Analysis , Aged , Biopsy , Carcinoma, Squamous Cell/genetics , Carcinoma, Squamous Cell/surgery , DNA, Neoplasm/genetics , Esophageal Neoplasms/genetics , Esophagectomy , Female , Humans , Male , Middle Aged , Neoplasm Metastasis , Neoplasm Staging , Preoperative Care , RNA, Neoplasm/genetics , Sensitivity and Specificity
10.
J Biochem ; 139(4): 697-703, 2006 Apr.
Article in English | MEDLINE | ID: mdl-16672270

ABSTRACT

We have developed a new DNA chip whose substrate has a unique minute columnar array structure made of plastic. The DNA chip exhibits ultrahigh sensitivity, up to 100-fold higher than that of reference DNA chips, which makes it possible to monitor gene expression profiles even with very small amounts of RNA (0.1-0.01 microg of total RNA) without amplification. Differential expression ratios obtained with the new DNA chip were validated against those obtained with quantitative real-time PCR assays. This novel microarray technology would be a powerful tool for monitoring gene expression profiles, especially for clinical diagnosis.


Subject(s)
Gene Expression Profiling/methods , Nucleic Acid Amplification Techniques/methods , Oligonucleotide Array Sequence Analysis/methods , RNA/metabolism , Gene Expression Profiling/instrumentation , Microscopy, Electron, Scanning , Nucleic Acid Hybridization/methods , Oligonucleotide Array Sequence Analysis/instrumentation , Plasminogen Activators/genetics , RNA/genetics , RNA, Messenger/genetics , RNA, Messenger/metabolism , Reproducibility of Results , Reverse Transcriptase Polymerase Chain Reaction/methods , Statistics, Nonparametric
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