DNA Microarray System Research Articles

The macrophage is a key factor in renal injuries caused by glomerular hyperfiltration.

Glomerular hyperfiltration is a common pathway leading to glomerulosclerosis in various kinds of kidney diseases. The 5/6 renal ablation is an established experimental animal model for glomerular hyperfiltration. On the other hand, low-grade inflammation is also a common mechanism for the progression of kidney diseases including diabetic nephropathy and atherosclerosis. Here we analyzed the gene expression profile in the remnant kidney tissues of 5/6 nephrectomized mice using a DNA microarray system and compared it with that of sham-operated control mice. The 5/6 nephrectomized mice showed glomerular hypertrophy and an increase in the extracellular matrix in the glomeruli. DNA microarray analysis indicated the up-regulated expression of various kinds of genes related to the inflammatory process in remnant kidneys. We confirmed the up-regulated expression of platelet factor-4, and monocyte chemoattractant protein-1, 2, and 5 in remnant kidneys by RT-PCR. The current results suggest that the inflammatory process is involved in the progression of glomerulosclerosis and is a common pathway of the pathogenesis of kidney disease.

Activation of the Interferon Pathway in Peripheral Blood of Patients with Sjögren’s Syndrome

DNA microarray analysis and quantitative real-time polymerase chain reaction (PCR) were performed to identify key target genes in peripheral blood from patients with Sjögren's syndrome (SS). DNA microarray analysis was performed in 19 patients with SS (all women) and 10 healthy controls (5 men and 5 women) using a low-density DNA microarray system with 778 genes. For confirmation, the expression of upregulated genes was analyzed by quantitative real-time PCR in another 37 SS patients (35 women and 2 men) and 9 healthy controls (8 women and 1 man). Relationships between gene signatures and various clinical measures, such as disease duration, symptoms and signs, complications, immunological findings, and salivary and lacrimal functions, were analyzed. Interferon-α (IFN-α)-inducible protein 27 (IFI27) showed the most significant difference between SS patients and controls in the microarray screening. We performed quantitative RT-PCR for IFI27. IFI27 gene expression level was increased in patients with SS compared with controls (p < 0.01) by real-time PCR, supporting our observations from the microarray data. The level of IFI27 was significantly correlated with serum IgG levels (r = 0.462, p < 0.01) and ß(2)-microglobulin (r = 0.385, p < 0.05), soluble interleukin 2 receptor (r = 0.473, p < 0.01), erythrocyte sedimentation rate (r = 0.333, p < 0.05), and antinuclear antibody titer (speckled pattern; r = 0.445, p < 0.01). Our results suggest that upregulation of IFN-inducible genes in SS patients is a systemic phenomenon, and IFN may play an important role in the pathogenesis of SS. The expression level of IFI27 could be an effective and specific biomarker associated with SS.

A DNA microarray for species identification of cetacean animals in Korean water

Some whales caught incidentally or illegally in Korean waters have been introduced into the seafood market. Thus, the Korea Coast Guard (KCG) monitors and reports incidentally caught whales. However, species identification of some carcasses based on morphological features is difficult and often inaccurate, particular for processed whale products in the marketplace. For these reasons, we developed a DNA microarray that distinguishes 9 species of cetacean animals for quick and simple species identification. We verified genetic variations in whales and designed species-specific oligonucleotide probes by sequence analysis of mitochondrial cytochrome c oxidase subunit I. Fluorescent dye-labeled DNA targets were generated with a fluorescent dye-labeled primer and hybridized onto a DNA microarray consisting of short probes that were 29-37 nucleotides in length. A DNA microarray system using species-specific probes was successful at rapidly identifying 9 species of whales on the basis of hybridization patterns.

Cytochrome P450 monooxygenases involved in anthracene metabolism by the white-rot basidiomycete Phanerochaete chrysosporium

Cytochrome P450 monooxygenases (P450s) involved in anthracene metabolism by the white-rot basidiomycete Phanerochaete chrysosporium were identified by comprehensive screening of both catalytic potentials and transcriptomic profiling. Functional screening of P. chrysosporium P450s (PcCYPs) revealed that 14 PcCYP species catalyze stepwise conversion of anthracene to anthraquinone via intermediate formation of anthrone. Moreover, transcriptomic profiling explored using a complementary DNA microarray system demonstrated that 12 PcCYPs are up-regulated in response to exogenous addition of anthracene. Among the up-regulated PcCYPs, five species showed catalytic activity against anthracene. Based upon both catalytic and transcriptional properties, these five species are most likely to play major roles in anthracene metabolic processes in vivo. Thus, the combination of functional screening and a microarray system may provide a novel strategy for obtaining a thorough understanding of the catalytic functions and biological impacts of PcCYPs.

An Event-Specific DNA Microarray To Identify Genetically Modified Organisms in Processed Foods

We developed an event-specific DNA microarray system to identify 19 genetically modified organisms (GMOs), including two GM soybeans (GTS-40-3-2 and A2704-12), thirteen GM maizes (Bt176, Bt11, MON810, MON863, NK603, GA21, T25, TC1507, Bt10, DAS59122-7, TC6275, MIR604, and LY038), three GM canolas (GT73, MS8xRF3, and T45), and one GM cotton (LLcotton25). The microarray included 27 oligonucleotide probes optimized to identify endogenous reference targets, event-specific targets, screening targets (35S promoter and nos terminator), and an internal target (18S rRNA gene). Thirty-seven maize-containing food products purchased from South Korean and US markets were tested for the presence of GM maize using this microarray system. Thirteen GM maize events were simultaneously detected using multiplex PCR coupled with microarray on a single chip, at a limit of detection of approximately 0.5%. Using the system described here, we detected GM maize in 11 of the 37 food samples tested. These results suggest that an event-specific DNA microarray system can reliably detect GMOs in processed foods.

Rapid detection of TEM, SHV and CTX-M extended-spectrum β-lactamases in Enterobacteriaceae using ligation-mediated amplification with microarray analysis

Fast and adequate detection of extended-spectrum beta-lactamases (ESBLs) is crucial for infection control measures and the choice of antimicrobial therapy. The aim of this study was to develop and evaluate a novel ESBL assay using ligation-mediated amplification combined with microarray analysis to detect the most prevalent ESBLs in Enterobacteriaceae: TEM, SHV and CTX-M. Analysis of the Lahey database revealed that the vast majority of TEM and SHV ESBLs differ from non-ESBL variants in three amino acid positions. TEM ESBLs have at least one of the following amino acid substitutions: R164S/H/C, G238D/N/S and E104K. In SHV ESBLs, one or more of the following substitutions is observed: D179A/N/G, G238S/A and E240K. Oligonucleotide probes were designed to detect these substitutions, covering 95% of ESBL TEM variants and 77% of ESBL SHV variants. In addition, probes were designed to distinguish between CTX-M groups 1, 2, 9 and 8/25. For evaluation of the assay, 212 Enterobacteriaceae isolates with various beta-lactamases were included (n = 106 ESBL positive). The sensitivity of the microarray was 101/106 (95%; 95% CI 89%-98%), and the specificity 100% (95% CI 97%-100%) using molecular characterization of ESBLs by PCR and sequencing as reference. Assay performance time was 8 h for 36 isolates. This novel commercially available DNA microarray system may offer an attractive option for rapid and accurate detection of CTX-M, TEM and SHV ESBL genes in Enterobacteriaceae in the clinical laboratory.

Comparison of three different methods for the detection of human papillomavirus infected in cervical lesions

e22166 Background: Several techniques are in use for HPV DNA detection: Hybrid Capture IIR(HCIIR) assay, PCR-based DNA microarray and Luminex-based assay. This study was aimed to examine the performances of three validated HPV assays. We compared the concordance between the three methods for detecting HPV in cervical specimen. Methods: Cervical samples of 189 Korean women (21∼77 ages, average 41.2 age) were tested for HPV detection in this study. We examined the performances of three validated HPV assays according to each method's instructions : 1) Hybrid Capture IIR (HCIIR) assay (HCIIR; Digene Corporation, USA) was used to test for detection of 18 types of HPV (high-risk types: HPV16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 68 and low-risk types types: HPV 6, 11, 42, 43, 44). 2) HPV DNA Chip, PCR-based DNA microarray system (MyGene Co., Seoul, South Korea) was used to test for genotyping of 24 types of HPV: 15 types of high-risk types (HPV 16, 18, 31, 33, 35, 39, 45, 51, 52, 53, 56, 58, 59, 66, 68) and 9 types of low-risk types (HPV6, 11, 34, 40, 42, 43, 44, 54, 70). 3) Luminex-based HPV test (YBT, Seoul, Korea) was performed for genotyping 26 types of HPV including 15 high-risk HPV types (HPV-16, 18, 31, 33, 35, 39, 45, 51, 52, 53, 56, 58, 59, 66, 68) and 11 low-risk types (HPV-6, 11, 34, 40, 42, 43, 44, 54, 55, 67, 70). Results: We compared HPV detection results obtained by the HC II assay with those from the Luminex assay. 51 cases were discordant: 29 samples were HC II negative/Luminex positive and 22 samples were HCII positive/Luminex negative. We also compared HPV detection results obtained by the DNA chip assay with those from the Luminex assay. 37 cases were discordant: 5 samples were DNA chip negative/Luminex positive and 32 samples were DNA chip positive/Luminex negative. Conclusions: The distribution of the HPV detection results obtained for 91 samples using three different methods suggested that the results of DNA chip assay are more often discordant (17.9% instead of 5.1% for Luminex assay and 5.1% for the HC II assay). No significant financial relationships to disclose.

Lipoic acid attenuates high fat diet-induced chronic oxidative stress and immunosuppression in mice jejunum: A microarray analysis

A high fat diet (HFD) has long been linked to immune dysfunction, including diminished numbers or reactivity of lymphocytes, increased susceptibility to infection, inhibited lymphocytes function during antigen-specific responses and developed oxidative stress. Whereas the molecular mechanistic events associated with immune deficiency remain to be fully determined. Using the DNA microarray system, we analyzed the gene expression patterns of lymphocyte related signal transduction proteins in jejunum of C57BL/6 mice in order to gain insight on the possible molecular mechanism by which HFD induced oxidative stress effects on signal transduction of lymphocytes. Results of present study showed that HFD induced oxidative stress and immunosuppression in jejunum. Antioxidant lipoic acid (LA) supplement ameliorated that HFD induced oxidative stress and immunosuppression by recovering transcriptional levels of the gene involved in B cell receptor, T cell differentiation signaling pathway, and free radical scavengers. The present study indicates that a HFD can induce chronic oxidative stress, suppress signal transduction of gut-associated lymphocytes, and lead to an inhibition of mucosal immunity.

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Application of DNA microarray technology to influenza A/Vietnam/1194/2004 (H5N1) vaccine safety evaluation

We propose that DNA microarray analysis can be used in the quality control of pandemic and endemic influenza vaccine. Based on the expression profiles of 76 genes in the rat lung one day after inoculation of influenza vaccine, we can distinguish whole-virion influenza vaccine (PDv: pandemic influenza vaccine and WPv: whole virion-particle vaccine) and sub-virion vaccine (HA vaccine) from saline. Among these 76 genes, we found genes up-regulated by influenza infection, as well as genes involved in the immune response, and interferon. Hierarchical clustering of each influenza vaccine by the expression profiles of these 76 genes matched data from current quality control tests in Japan, such as the abnormal toxicity test (ATT) and the leukopenic toxicity test (LTT). Thus, it can be concluded that DNA microarray technology is an informative, rapid and highly sensitive method with which to evaluate the quality of influenza vaccines. Using DNA microarray system, consistent with the results of the ATT and LTT, it was clarified that there was no difference in vaccine quality between PDv and WPv.

Quantitative oligonucleotide microarray data analysis with an artificial standard probe strategy

Quantitative data analysis is an important element in several applications of DNA microarray, including mRNA expression profiling and estimation of infectious doses for pathogens. Here, we introduce an artificial standard probe strategy for quantitative pathogen detection using an oligonucleotide chip as a model system. The standard capture probe sequence was artificially designed to prevent non-specific hybridization with bacterial targets. Based on the fluorescence intensities of artificial standard spots, the raw fluorescence intensity data for specific spots could be corrected to generate linear correlations with target concentrations. Therefore, our novel artificial standard probe may be effectively applied for the correction of chip-to-chip variations and quantitative data analysis of a one-color labeled DNA microarray system.

Development of a photodiode array biochip using a bipolar semiconductor and its application to detection of human papilloma virus

We describe a DNA microarray system using a bipolar integrated circuit photodiode array (PDA) chip as a new platform for DNA analysis. The PDA chip comprises an 8 x 6 array of photodiodes each with a diameter of 600 microm. Each photodiode element acts both as a support for an immobilizing probe DNA and as a two-dimensional photodetector. The usefulness of the PDA microarray platform is demonstrated by the detection of high-risk subtypes of human papilloma virus (HPV). The polymerase chain reaction (PCR)-amplified biotinylated HPV target DNA was hybridized with the immobilized probe DNA on the photodiode surface, and the chip was incubated in an anti-biotin antibody-conjugated gold nanoparticle solution. The silver enhancement by the gold nanoparticles bound to the biotin of the HPV target DNA precipitates silver metal particles at the chip surfaces, which block light irradiated from above. The resulting drop in output voltage depends on the amount of target DNA present in the sample solution, which allows the specific detection and the quantitative analysis of the complementary target DNA. The PDA chip showed high relative signal ratios of HPV probe DNA hybridized with complementary target DNA, indicating an excellent capability in discriminating HPV subtypes. The detection limit for the HPV target DNA analysis improved from 1.2 nM to 30 pM by changing the silver development time from 5 to 10 min. Moreover, the enhanced silver development promoted by the gold nanoparticles could be applied to a broader range of target DNA concentration by controlling the silver development time.

Evaluation of the Premi®Test Salmonella, a commercial low-density DNA microarray system intended for routine identification and typing of Salmonella enterica

A new commercial system based on genetic profiling and aimed at identifying Salmonella enterica serovars was evaluated by comparing its performance with classical serotyping on 443 strains. Within 62 serovars represented, 60 gave unique genetic profiles while 2 were undistinguishable. Results were obtained within 8 h, were reproducible and clear-cut. The system allowed single-tube processing of the samples and required no peculiar technical skill. It showed interesting potential for routine laboratory testing.

Expression profile of liver genes in response to hepatotoxicants identified using a SAGE-based customized DNA microarray system

Gene expression analysis using customized or focused DNA microarrays is favorable because of a reduction in the cost and time needed for the analysis. To examine the effect of chemicals on the liver, we developed an in-house cDNA microarray system, mouse Liver Stress Array ver. 1.0, containing 355 unique genes involved in drug metabolism, inflammation and liver-related proteins. These genes were selected for sensing the homeostasis of the liver and based on the information of liver transcriptome revealed by serial analysis of gene expression. By using this customized microarray, we analyzed gene expression changes in the mouse liver treated by 11 known hepatotoxicants. Gene expression measurements corresponding to the in vivo response to known hepatotoxicants revealed that profiles of chemicals with similar mechanisms clustered together. For each of the chemicals tested, several genes that were induced or repressed were common in each chemical exposure, whereas other genes were unique for the specific class compound. Ingenuity pathways analysis revealed that significant alterations in gene expression occurred in a number of biological networks by these treatments. Although the genes spotted on our array was limited to a highly focused set for toxicity classification, this work provides proof of concept that patterns of gene regulation assessed by a focused array system are useful to classify unknown chemicals.

Enhanced Expression of Asparagine Synthetase under Glucose-Deprived Conditions Protects Pancreatic Cancer Cells from Apoptosis Induced by Glucose Deprivation and Cisplatin

Although hypovasculature is an outstanding characteristic of pancreatic cancers, the tumor cells survive and proliferate under severe hypoxic, glucose-deprived conditions caused by low blood supply. It is well known that the hypoxia-inducible factor-1 pathway is essential for the survival of pancreatic cancer cells under hypoxic conditions. To discover how pancreatic cancer cells adapt to glucose deprivation as well as hypoxia, we sought glucose deprivation-inducible genes by means of a DNA microarray system. We identified 63 genes whose expression was enhanced under glucose-deprived conditions at >2-fold higher levels than under normal glucose conditions. Among these genes, asparagine synthetase (ASNS) was studied in detail. Although it is known to be associated with drug resistance in leukemia and oncogenesis triggered by mutated p53, its function is yet to be determined. In this study, we found that glucose deprivation induced the overexpression of ASNS through an AMP-activated protein kinase-independent and activating transcription factor-4-dependent manner and that ASNS protects pancreatic cancer cells from apoptosis induced by glucose deprivation itself. ASNS overexpression also induced resistance to apoptosis triggered by cisplatin [cis-diammine-dichloroplatinum (CDDP)] and carboplatin, but not by 5-fluorouracil, paclitaxel, etoposide, or gemcitabine. We show that glucose deprivation induces the activation of c-jun NH(2)-terminal kinase (JNK)/stress-activated protein kinase (SAPK) in a mock transfectant but not in an ASNS transfectant. Consequently, an inhibitor of JNK/SAPK decreased the sensitivity of pancreatic cancer cells to apoptosis by glucose deprivation and CDDP. These results strongly suggest that ASNS is induced by glucose deprivation and may play a pivotal role in the survival of pancreatic cancer cells under glucose-deprived conditions.

Changes of gene expression profiles in macrophages stimulated by angiotensin II — Angiotensin II induces MCP-2 through AT1-receptor

Macrophages play critical roles in the development of atherosclerosis and diabetic nephropathy as well as many inflammatory diseases. Angiotensin II type 1 receptor antagonists (AIIA) are beneficial for the prevention of atherosclerosis and diabetic nephropathy suggesting that angiotensin II (Ang II) promotes the development of these diseases. It has recently been reported that Ang II exerts proinflammatory actions in vivo and in vitro. This study was aimed to clarify the direct effects of Ang II on monocytes/macrophages. PMA-treated THP-1 cells, a human monocytic leukaemia cell line, were treated with Ang II (10-6 mol/L) for 24 hours with or without AIIA (CV11974). We evaluated gene expression profiles of these cells using DNA microarray system and quantified them by real-time RT-PCR. DNA microarray revealed that in total 19 genes, including monocyte chemoattractant protein (MCP)-2, were up-regulated by Ang II and down-regulated by AIIA. Real-time RT-PCR showed that up-regulation of MCP-2 with Ang II is blocked by the AIIA (CV11974) but not by an AT2-receptor antagonist. These results suggest that Ang II directly stimulates MCP-2 expression through AT1-receptors in activated macrophages. Ang II may contribute to the persistence or amplification of microinflammation in vessel walls, heart and kidney. Vasculoprotective or renoprotective effects of AIIA might partly depend on direct anti-inflammatory effects on macrophages.

Modulatory effects of Echinacea purpurea extracts on human dendritic cells: A cell- and gene-based study

Echinacea spp. are popularly used as an herbal medicine or food supplement for enhancing the immune system. This study shows that plant extracts from root [R] and stem plus leaf [S+L] tissues of E. purpurea exhibit opposite (enhancing vs inhibitory) modulatory effects on the expression of the CD83 marker in human dendritic cells (DCs), which are known as professional antigen-presenting cells. We developed a function-targeted DNA microarray system to characterize the effects of phytocompounds on human DCs. Down-regulation of mRNA expression of specific chemokines (e.g., CCL3 and CCL8) and their receptors (e.g., CCR1 and CCR9) was observed in [S+L]-treated DCs. Other chemokines and regulatory molecules (e.g., CCL4 and CCL2) involved in the c-Jun pathway were found to be up-regulated in [R]-treated DCs. This study, for the first time, demonstrates that E. purpurea extracts can modulate DC differentiation and expression of specific immune-related genes in DCs.

SI35 Blood Group Antigen Typing Using DNA Microarray BeadChipsTM–First UK Report

Background The determination of human blood group phenotypes has routinely involved red cell agglutination techniques. The genes for all but one of the 29 human blood group systems have now been cloned and the molecular bases of all clinically significant blood group polymorphisms are known. Consequently these DNA sequences can be used to predict blood group phenotypes. Molecular typing as an adjunct to serology offers improved accuracy and can save both rare reagents and laboratory time while screening for compatible donor units. This study determined the feasibility of analysis of single nucleotide polymorphisms (SNPs) associated with a variety of blood group antigens using a DNA microarray system with a beadchip technology platform.Method SNPs were tested by elongation‐mediated analysis of polymorphisms (eMAPTM) using allele‐specific oligonucleotides with variable 3’‐terminal sequences attached to colour‐encoded beads. The beads are assembled into arrays on semiconductor chips (BeadChipTM), available from Bioarray Solutions. Elongation products for SNPs or small deletions/insertions were simultaneously detected by instant imaging of fluorescence signals from the entire array. Seventeen polymorphisms associated with Human Erythrocyte Antigens and one mutation associated with hemoglobinopathies (CO, DI, DO, FY, JK, KEL, LU, LW, MNS, SC and HgbS) were included in the HEA BeadChipTM Kit.Results The system was validated using a reference DNA panel of known phenotype (n = 30) and genotype, established by conventional DNA‐PCR SSP assays. Replicate testing (n = 6 reps for eight samples) gave 100% consistent data demonstrating the Bioarray system to be both reliable and reproducible. The initial concordance rate with serology was 99.2%.Conclusion The BeadChip System represents the first integrated flexible array solution for high complexity transfusion testing, accommodating ‘multiplex’ analysis of polymorphisms. This high throughput, microarray system offers the potential to determine extended genotypes of patients and maintain an extended‐genotype, national panel of donors to provide compatible blood for sensitised patients. The initial high concordance rate and the rarity of CE marked phenotyping reagents justifies its further development.

Interrogating genomic diversity of E. coli O157:H7 using DNA tiling arrays

Here, we describe a novel microarray-based approach for investigating the genomic diversity of Escherichia coli O157:H7 in a semi-high throughput manner using a high density, oligonucleotide-based microarray. This microarray, designed to detect polymorphisms at each of 60,000 base-pair (bp) positions within an E. coli genome, is composed of overlapping 29-mer oligonucleotides specific for 60 equally spaced, 1000-bp loci of the E. coli O157:H7 strain EDL933 chromosome. By use of a novel 12-well microarray that permitted the simultaneous investigation of 12 strains, the genomes of 44 individual isolates of E. coli O157:H7 were interrogated. These analyses revealed more than 150 single nucleotide polymorphisms (SNPs) and several deletions and amplifications in the test strains. Pyrosequencing was used to confirm the usefulness of the novel SNPs by determining their allelic frequency among a collection of diverse isolates of E. coli O157:H7. The tiling DNA microarray system would be useful for the tracking and identification of individual strains of E. coli O157:H7 needed for forensic investigations.

DNA microarray analysis for the detection of mutations in hemophilia A.

Congenital deficiency of factor (F) VIII results in the inherited X-linked bleeding disorder hemophilia A. More than 900 different mutations are reported in the hemophilia A mutation database with the largest number of mutations being single nucleotide substitutions distributed throughout the gene. Complicating the molecular characterization of this disease is the complexity of the F8 gene, the mutational heterogeneity, and technical limitations of the current mutation detection techniques. Development of a DNA oligonucleotide microarray-based technique for F8 gene analysis to detect hemophilia A mutations. To construct the oligonucleotide DNA microarray system: a total of 720, one base pair overlapping, 25-mer perfect match probes were designed from six exons of the F8 gene. Twenty-two different F8 gene mutations previously identified by CSGE and DNA sequence analysis were tested by using a loss-of-signal analysis approach. Differentially labeled wild type and hemophilic samples were co-hybridized to the array. Sequence alterations were detected by quantifying relative losses of test sample hybridization signals to the perfectly matched probes. A total of 22 different F8 mutations were tested. To test the sensitivity of the system, a blinded study was performed on 16 of the samples. F8 gene mutations can be detected with 96% efficiency with this microarray system. This proof-of-principle study has demonstrated that a F8 DNA microarray platform is an alternative gene mutation analysis approach that has a high sensitivity, and reproducibility. The methodology is, however, expensive and time consuming, and with the reduction in sequencing costs, direct sequencing is now the most cost and time efficient strategy for hemophilia A mutation analysis.