DNA Oligonucleotide Arrays Research Articles

Polymer Pen Lithography-Fabricated DNA Arrays for Highly Sensitive and Selective Detection of Unamplified Ganoderma Boninense DNA.

There is an increasing demand for lithography methods to enable the fabrication of diagnostic devices for the biomedical and agri-food sectors. In this regard, scanning probe lithography methods have emerged as a possible approach for this purpose, as they are not only convenient, robust and accessible, but also enable the deposition of “soft” materials such as complex organic molecules and biomolecules. In this report, the use of polymer pen lithography for the fabrication of DNA oligonucleotide arrays is described, together with the application of the arrays for the sensitive and selective detection of Ganoderma boninense, a fungal pathogen of the oil palm. When used in a sandwich assay format with DNA-conjugated gold nanoparticles, this system is able to generate a visually observable result in the presence of the target DNA. This assay is able to detect as little as 30 ng of Ganoderma-derived DNA without any pre-amplification and without the need for specialist laboratory equipment or training.

Detection and Genotyping of Campylobacter jejuni and Campylobacter coli by Use of DNA Oligonucleotide Arrays

Campylobacter have emerged as the most common bacterial food-borne illness in the developed world. The ability to reduce Campylobacter infections in humans is linked to the full comprehension of the principal key aspects of its infection cycle. A microbial diagnostic microarray detecting Campylobacter housekeeping, structural, and virulence associated genes was designed and validated using genomic DNA from reference and field strains of Campylobacter jejuni and coli isolated from human, chicken, and raw milk. This microarray was confirmed to be a powerful diagnostic tool for monitoring emerging Campylobacter pathotypes as well as for epidemiological, environmental, and phylogenetic studies including the evaluation of genome plasticity.

Robust Gene Expression Index

The frequentist gene expression index (FGX) was recently developed to measure expression on Affymetrix oligonucleotide DNA arrays. In this study, we extend FGX to cover nonnormal log expressions, specifically long‐tailed symmetric densities and call our new index as robust gene expression index (RGX). In estimation, we implement the modified maximum likelihood method to unravel the elusive solutions of likelihood equations and utilize the Fisher information matrix for covariance terms. From the analysis via the bench‐mark datasets and simulated data, it is shown that RGX has promising results and mostly outperforms FGX in terms of relative efficiency of the estimated signals, in particular, when the data are nonnormal.

Monitoring tumour progression in a patient with colorectal carcinoma using comparative genomic hybridisation on DNA oligonucleotide arrays (aCGH)

Aims: Genetic heterogeneity is a hallmark of cancer and evolves during tumour development as well as disease progression and metastasis expansion. Revealing genetic changes in the primary tumour and the autologous metastases of cancer patients is an important step towards targeted therapies. Recent studies have shown enormous differences in genetic constitution between primary tumour and metastases. This fact could be a reason for failure of currently used therapies, which are fitted to the genetic aberrations found in the primary tumour.

Gene expression profiles predicting the response to IFN-β and a combination of temozolomide and IFN-β in malignant gliomas

Temozolomide (TMZ) is an alkylating agent that has yielded significant benefits and is a current standard agent in the treatment of malignant gliomas. However, its survival benefit remains unsatisfactory. Recently, a synergistic antitumor effect between TMZ and interferon-β (IFN-β) was reported in malignant glioma cells. The Japan Clinical Oncology Group (JCOG) brain tumor study group has recently began a randomized phase II study to evaluate the clinical effectiveness of combination therapy with TMZ and IFN-β in glioblastomas. However, it is not sufficient just to evaluate the mechanisms and establish an experimental basis for rational clinical therapy with IFN-β and TMZ. The precise mechanisms governing the direct effects of IFN-β and a combination of IFN-β and TMZ in gliomas are not yet fully understood. To gain insight into the mechanisms of sensitivity/resistance involving IFN-β and combination therapy with IFN-β and TMZ, and further to identify new marker(s) that could be used clinically to predict the response to such therapy and new target gene(s) for therapies related to malignant glioma patho-genesis, we evaluated the gene expression profiles of human malignant glioma cell lines employing a high-density oligo-nucleotide DNA array, GeneChip. We present a list of the most highly upregulated and downregulated genes which may be involved in conferring a response to IFN-β and synergistic effect between IFN-β and TMZ in malignant gliomas. Although the present study has several limitations, our reported candidate genes could represent not only potential molecular markers but also chemotherapy targets for improving the treatment outcome by devising strategies that are able to circumvent primary drug resistance in malignant gliomas.

Development-dependent changes in the tight DNA-protein complexes of barley on chromosome and gene level

BackgroundThe tightly bound to DNA proteins (TBPs) is a protein group that remains attached to DNA with covalent or non-covalent bonds after its deproteinisation. The functional role of this group is as yet not completely understood. The main goal of this study was to evaluate tissue specific changes in the TBP distribution in barley genes and chromosomes in different phases of shoot and seed development. We have: 1. investigated the TBP distribution along Amy32b and Bmy1 genes encoding low pI α-amylase A and endosperm specific β-amylase correspondingly using oligonucleotide DNA arrays; 2. characterized the polypeptide spectrum of TBP and proteins with affinity to TBP-associated DNA; 3. localized the distribution of DNA complexes with TBP (TBP-DNA) on barley 1H and 7H chromosomes using mapped markers; 4. compared the chromosomal distribution of TBP-DNA complexes to the distribution of the nuclear matrix attachment sites.ResultsIn the Amy32b gene transition from watery ripe to the milky ripeness stage of seed development was followed by the decrease of TBP binding along the whole gene, especially in the promoter region and intron II. Expression of the Bmy1 gene coupled to ripening was followed by release of the exon III and intron III sequences from complexes with TBPs. Marker analysis revealed changes in the association of chromosome 1H and 7H sites with TBPs between first leaf and coleoptile and at Zadoks 07 and Zadoks 10 stages of barley shoot development. Tight DNA-protein complexes of the nuclear matrix and those detected by NPC-chromatography were revealed as also involved in tissue- and development-dependent transitions, however, in sites different from TBP-DNA interactions. The spectrum of TBPs appeared to be organ and developmental-stage specific. Development of the first leaf and root system (from Zadoks 07 to Zadoks 10 stage) was shown as followed by a drastic increase in the TBP number in contrast to coleoptile, where the TBPs spectrum became poor during senescence. It was demonstrated that a nuclear protein of low molecular weight similar to the described TBPs possessed a high affinity to the DNA involved in TBP-DNA complexes.ConclusionPlant development is followed by redistribution of TBP along individual genes and chromosomes.

Transcription- and Apoptosis-Dependent Long-Range Distribution of Tight DNA–Protein Complexes in the Chicken α-Globin Gene

The proteins tightly bound to DNA (TBP) are a group of proteins that remain attached to DNA with covalent or noncovalent bonds after its deproteinization, and have been hypothesized to be involved in regulation of gene expression. To investigate this question further, oligonucleotide DNA arrays were used to determine the distribution of tightly bound proteins along a 100-kb DNA fragment surrounding the chicken alpha-globin gene domain in DNA from chicken erythrocytes, liver, and AEV-transformed HD3 (erythroblast) cells in different physiological conditions. DNA was fractionated into TBP-free (F) and TBP-enriched (R) fractions by separation on nitrocellulose, and these fractions were used as probes for hybridization with the microarray. In erythrocytes, the site 60 kb from the 5' end of the sequence and containing a LINE family CR1 repeat was TBP enriched, but in HD3 cells this sequence was devoid of TBPs. Thus cessation of transcription of the domain is followed by an F-R transition of this site. In apoptotic HD3 cells, TBPs remained attached to DNA only at a site situated 16 kb from the 5' end of the sequence. These data confirm and extend previous conclusions about the specificity of the DNA sequences that preferably form tight complexes with proteins and about the differentiation-specific distribution of the TBPs in different cell lineages. Binding of TBPs appears to be independent of primary DNA sequence.

Improved Elucidation of Biological Processes Linked to Diabetic Nephropathy by Single Probe-Based Microarray Data Analysis

BackgroundDiabetic nephropathy (DN) is a complex and chronic metabolic disease that evolves into a progressive fibrosing renal disorder. Effective transcriptomic profiling of slowly evolving disease processes such as DN can be problematic. The changes that occur are often subtle and can escape detection by conventional oligonucleotide DNA array analyses.Methodology/Principal FindingsWe examined microdissected human renal tissue with or without DN using Affymetrix oligonucleotide microarrays (HG-U133A) by standard Robust Multi-array Analysis (RMA). Subsequent gene ontology analysis by Database for Annotation, Visualization and Integrated Discovery (DAVID) showed limited detection of biological processes previously identified as central mechanisms in the development of DN (e.g. inflammation and angiogenesis). This apparent lack of sensitivity may be associated with the gene-oriented averaging of oligonucleotide probe signals, as this includes signals from cross-hybridizing probes and gene annotation that is based on out of date genomic data. We then examined the same CEL file data using a different methodology to determine how well it could correlate transcriptomic data with observed biology. ChipInspector (CI) is based on single probe analysis and de novo gene annotation that bypasses probe set definitions. Both methods, RMA and CI, used at default settings yielded comparable numbers of differentially regulated genes. However, when verified by RT-PCR, the single probe based analysis demonstrated reduced background noise with enhanced sensitivity and fewer false positives.Conclusions/SignificanceUsing a single probe based analysis approach with de novo gene annotation allowed an improved representation of the biological processes linked to the development and progression of DN. The improved analysis was exemplified by the detection of Wnt signaling pathway activation in DN, a process not previously reported to be involved in this disease.

Detection of Genetically Modified Canola Using Multiplex PCR Coupled with Oligonucleotide Microarray Hybridization

A rapid method was developed for concurrent screening of transgenic elements in GM canola. This method utilizes a single multiplex PCR coupled with an oligonucleotide DNA array capable of simultaneously detecting the 12 approved GM canola lines in Canada. The assay includes construct-specific elements for identification of approved lines, common elements (e.g., CaMV 35S promoter, Agrobacterium tumefaciens nos terminator, or nptII gene) for screening of approved or unapproved lines, a canola-specific endogenous gene, and endogenous genes from heterologous crops to serve as additional controls. Oligonucleotide probes were validated individually for functionality and specificity by amplification of specific transgene sequences from appropriate GM canola lines corresponding to each probe sequence, and hybridization of amplicons to the array. Each target sequence hybridized to its corresponding oligonucleotide probe and no significant cross-hybridization was observed. The limit of detection was examined for the GM lines GT73, T45, and MS8/RF3, and was determined to be 0.1%, 0.1%, and 0.5%, respectively, well within the European food and feed labeling threshold level of 0.9% for approved GM product. Practically, the method was demonstrated to be effective for the detection of GM canola in several types of animal feed, as well as in commercial canola meal.

Identification of a Cigarette Smoke–Responsive Region in the Distal MMP-1 Promoter

Tobacco-related diseases are leading causes of death worldwide, and many are associated with expression of matrix metalloproteinase-1 (MMP-1). We have reported extracellular signal-regulated kinase (ERK)1/2-dependent induction of MMP-1 by cigarette smoke in lung epithelial cells. Our objectives were to define regions of the human MMP-1 promoter required for activation by smoke, to identify differences in responses of the 1G/2G -1607 polymorphic promoters to smoke, and to identify relevant transcription factors whose activity in airway epithelial cells is increased by smoke. The responses of deletion and mutant promoter constructs were measured in transfected cells during exposure to cigarette smoke extract (CSE). DNA oligonucleotide arrays were used to identify transcription factors activated after smoke exposure. CSE activated the MMP-1 promoter, and this induction was prevented by PD98059 blockade of ERK1/2 phosphorylation. Deletion studies revealed the distal 1kb promoter region (-4438 to -3280 upstream of the transcription start site) is essential for CSE induction of MMP-1, and confers activation of a minimal promoter. Studies of 1G and 2G MMP-1 polymorphic promoter variants revealed higher 2G allele basal and CSE-responsive activities than the 1G allele. Cotransfection, mithramycin, and electrophoretic mobility shift assay studies identified activating and repressive roles for Sp1 and PEA3 transcription factors, respectively. Oligonucleotide DNA arrays confirmed activation of Sp1 and PEA3 by CSE. These data demonstrate that the MMP-1 promoter is a direct target of cigarette smoke in lung epithelial cells. This characterization of a smoke response region in the distal MMP-1 promoter has implications for smoking-related diseases such as cancer, heart disease, and emphysema.

Differential gene expression profiles in acute hepatic failure model in mice infected with MHV-3 virus intervened by anti-hepatic failure compound

Differential gene expression profiles in Balb/cJ mouse model of acute hepatic failure infected with MHV-3 virus intervened by anti-hepatic failure compound (AHFC) and the changes of cytokines regulated by genes were investigated. The Balb/cj mice were divided into AHFC-intervened group and control group randomly. Acute hepatic failure model of Balb/cJ mice infected with MHV-3 virus was established. The survival rate in the two groups was observed. It was found that the survival rate in the AHFC-intervened group and control group was 90% and 50% respectively 48 h after intraperitoneal injection of MHV-3 (P<0.05). Before and after the experiment, the cytokines in peripheral blood of the survival mice were determined, and RNA was extracted from survival mouse liver tissue for the analysis of the differential gene expression by a 36 kb mouse oligonucleotide DNA array. In all the genes of microarray there were 332 genes expressed differently in the two groups, in which 234 genes were up-regulated and 78 genes down-regulated. Through clustering analysis, the differential expression of immune related genes, including TNF receptor superfamily, Kctd9, Bcl-2, Fgl2, IL-8, IL-6, IFN-gamma, TNF-alpha etc. might be related with the curative effectiveness of AHFC. It was suggested that AHFC can balance the immune state of mouse model of acute hepatic failure infected with MHV-3 virus mainly through regulating the expression of immune related genes, decrease the immune damage and inhibit liver cell apoptosis of mouse acute hepatic failure model obviously so as to increase the survival rate of mouse models of acute hepatic failure.

Detection and Genotyping of Arcobacter and Campylobacter Isolates from Retail Chicken Samples by Use of DNA Oligonucleotide Arrays

To explore the use of DNA microarrays for pathogen detection in food, we produced DNA oligonucleotide arrays to simultaneously determine the presence of Arcobacter and the presence of Campylobacter in retail chicken samples. Probes were selected that target housekeeping and virulence-associated genes in both Arcobacter butzleri and thermotolerant Campylobacter jejuni and Campylobacter coli. These microarrays showed a high level of probe specificity; the signal intensities detected for A. butzleri, C. coli, or C. jejuni probes were at least 10-fold higher than the background levels. Specific identification of A. butzleri, C. coli, and C. jejuni was achieved without the need for a PCR amplification step. By adapting an isolation method that employed membrane filtration and selective media, C. jejuni isolates were recovered from package liquid from whole chicken carcasses prior to enrichment. Increasing the time of enrichment resulted in the isolation of A. butzleri and increased the recovery of C. jejuni. C. jejuni isolates were further classified by using an additional subset of probes targeting the lipooligosaccharide (LOS) biosynthesis locus. Our results demonstrated that most of the C. jejuni isolates likely possess class B, C, or H LOS. Validation experiments demonstrated that the DNA microarray had a detection sensitivity threshold of approximately 10,000 C. jejuni cells. Interestingly, the use of C. jejuni sequence-specific primers to label genomic DNA improved the sensitivity of this DNA microarray for detection of C. jejuni in whole chicken carcass samples. C. jejuni was efficiently detected directly both in package liquid from whole chicken carcasses and in enrichment broths.

Profiling of genes differentially expressed in a rat of early and later gestational ages with high‐density oligonucleotide DNA array

The early gestational fetus heals dermal wounds rapidly and scarlessly. This phenomenon appears to be intrinsic to fetal skin and is probably modulated by interplay of many genes. We ventured to study differences in gene expression between earlier gestational skin (EGS) and later gestational skin (LGS) with the aid of high-density oligonucleotide DNA array to explore the molecular mechanism underlying scarless healing. Total RNA was isolated from fetal Wistar rat skin of the scarless (E15) and scar-forming (E18) periods of gestation (term=21.5 days), and purified to mRNAs. Both the mRNAs from EGS and LGS were reversely transcribed to cDNAs, and were labeled with the incorporation of fluorescent dCTP for preparing the hybridization probes through single primer amplification reaction and Klenow labeling methods. The mixed probes were then hybridized to the oligonucleotide DNA arrays that contained 5,705 DNA fragments representing 5,705 rat genes. After highly stringent washing, the microarray was scanned for fluorescent signals to display the differentially expressed genes between two groups of tissues. Among 5,705 rat genes, there were 53 genes (0.93%) with differentially expressed levels between EGS and LGS; 27 genes, including fibroblast growth factor 8 and follistatin, were up-regulated (0.47%); and 26 genes, containing lymphoid enhancer binding factor-1 and beta-catenin, were down-regulated (0.46%) in fetal skin of scarless period vs. scar-forming period. Analyses of genes related to ion channels, growth factors, extracellular matrix and cellular skeleton, and movement confirmed that our molecular data obtained by oligonucleotide DNA array were consistent with the published biochemical and clinical findings of fetal scarless healing. Stronger expression of fibroblast growth factor 8, follistatin, and weaker expression of lymphoid enhancer binding factor-1 and beta-catenin in EGS vs. LGS were also testified with reverse transcription-polymerase chain reaction and Western blotting methods. Oligonucleotide DNA array was a powerful tool for investigating different gene expression between scarless and scar-forming periods of gestation in the rat fetal skin. Many genes were involved in the phenotypic transition from scarless to scar-forming wound repair during gestation. Further analysis of the obtained genes will help to understand the molecular mechanism of fetal scarless healing.

Genome-wide detection of human copy number variations using high-density DNA oligonucleotide arrays

Recent reports indicate that copy number variations (CNVs) within the human genome contribute to nucleotide diversity to a larger extent than single nucleotide polymorphisms (SNPs). In addition, the contribution of CNVs to human disease susceptibility may be greater than previously expected, although a complete understanding of the phenotypic consequences of CNVs is incomplete. We have recently reported a comprehensive view of CNVs among 270 HapMap samples using high-density SNP genotyping arrays and BAC array CGH. In this report, we describe a novel algorithm using Affymetrix GeneChip Human Mapping 500K Early Access (500K EA) arrays that identified 1203 CNVs ranging in size from 960 bp to 3.4 Mb. The algorithm consists of three steps: (1) Intensity pre-processing to improve the resolution between pairwise comparisons by directly estimating the allele-specific affinity as well as to reduce signal noise by incorporating probe and target sequence characteristics via an improved version of the Genomic Imbalance Map (GIM) algorithm; (2) CNV extraction using an adapted SW-ARRAY procedure to automatically and robustly detect candidate CNV regions; and (3) copy number inference in which all pairwise comparisons are summarized to more precisely define CNV boundaries and accurately estimate CNV copy number. Independent testing of a subset of CNVs by quantitative PCR and mass spectrometry demonstrated a >90% verification rate. The use of high-resolution oligonucleotide arrays relative to other methods may allow more precise boundary information to be extracted, thereby enabling a more accurate analysis of the relationship between CNVs and other genomic features.

MiRNA Expression Signatures in Acute Myeloid Leukemia Are Predictors for Patient Outcome.

Only recently, a class of gene regulatory molecules (miRNA's) has been described that have the potential for posttranscriptional and translational gene regulation. MiRNA's can influence critical cellular properties like proliferation and differentiation. Since these properties vary substantially in genetically defined AML subsets we have chosen to analyze AML patients with inv(16) (n=12); standard risk cytogenetic classification (mainly comprising the normal karyotype) (SR) (n=21) and with monosomy 7 or deletion7q (n=17) using DNA oligonucleotide arrays which are employing the mirVANA miRNA probe set. Principal Component Analysis (PCA) of AML samples from patients with inv(16) showed only minor differences to normal bone marrow (BM). In contrast AML patients with SR could be clearly divided in two subgroups - one subgroup that showed miRNA expression pattern close to BM and a distinctly different subgroup. Comparison of SR patient samples with BM using ANOVA tests revealed 9 differentially expressed miRNA's (mir-223; mir-15a; mir-16; mir-203; mir-103; mir-23b; mir-107, mir-17–5p and mir23a). AML patients with aberrations of chromosome 7 showed highly distinct PCA pattern as compared to BM samples. 64 miRNA's were found to be differentially expressed in those patients as compared to BM. All miRNA's located on chromosome7 that were included in the array were found downregulated in AML patients with aberrant chromosome 7 (e.g.mir-335).To further substantiate the microarray data we analyzed a total of 108 AML patients with inv(16), SR cytogenetics and aberrations of chromosome 7 for mir-23a, mir-223, mir-16 and mir-335 expression by qRT-PCR. Supporting the microarray data it could be shown that the median expression levels of mir-23a, mir-223 and mir-16 were lowest in patients with SR cytogenetics, whereas AML patients with chromosome 7 aberrations showed lowest median expression for mir-335 transcripts. This was in clear contrast to the expression of mir-150, a miRNA which could be previously shown to associate with immature T-cells and which had the highest expression in AML samples with chromosome 7 aberrations. Expression of mir-150 was correlated with a high CD34 percentage in the investigated AML samples (p<0.001) whereas mir-223 did not correlate with CD34 but was strongly associated with the surface expression of CD14 (p<0.001). In an in-vitro model of G-CSF induced CD34+ stem cell differentiation mir-223 and mir-16 were slightly upregulated whereas mir-150 disappeared during the process of differentiation. Finally, the investigated miRNA were entered in a model for outcome prediction in AML samples with SR cytogenetics including known risk factors. Within this model the ratio of mutant vs. wt FLT-3 was the strongest predictor of overall survival (p<0.01). Additonally, mir-23a expression proved to be an independent negative prognostic factor for AML patients with SR cytogenetics (p<0.05).In conclusion, the presented data demonstrate specific expression profiles of miRNA's in AML patients with different cytogenetic risk profiles. As it could be shown for patients with SR cytogenetics the observed expression profiles are likely to contribute to a deregulated differentiation and may influence therapeutic outcome.

Curcumin Inhibits Proliferation of Leukemic Cells Expressing P210BCR-ABL or BCR-ABL-T315I and Prolongs Survival of Mice with B-Cell Acute Lymphoblastic Leukemia (B-ALL).

The prognosis of BCR-ABL-induced B-ALL remains dismal with no effective chemotherapies currently available. Curcumin, a natural compound derived from tumeric, has been shown to induce apoptosis in the leukemic cell line K562 and in many solid tumor cells. We tested whether curcumin has an inhibitory effect on B-lymphoid transformation by wild type BCR-ABL or BCR-ABL-T315I mutant. We first used BCR-ABL-expressing pre-B cells isolated from B-ALL mice and BCR-ABL-expressing BaF/3 cells. We treated these cells with different concentrations of curcumin (10, 30, or 50 uM), and found that proliferation of the cells was significantly inhibited at concentrations as low as 10 uM. Curcumin also induced apoptosis of the cells, as shown by increased Annexin V-positive cells detected by FACS. Western blot analysis showed that Curcumin treatment resulted in decreased JNK2 activation and a significant increase in IKKa/b expression. To further investigate the underlying mechanisms for cellular apoptosis induced by curcumin, we screened for apoptosis related transcription factors that were activated or inactivated in curcumin-treated BCR-ABL-expressing pre-B cells isolated from B-ALL mice. To do so, we probed nuclear extracts of these cells onto a commercial DNA oligonucleotide arrays (Panomics) representing the canonical binding sites for 52 transcription factors. We observed decreased expression of NFkB at 48 hours and a significant increase in expression of AP-2, p53, and vitamin D receptor 3 (VDR3) at 24 hours. The p53 induction by curcumin was further demonstrated by Western blotting. To examine the efficacy of curcumin on B-ALL, we induced B-ALL in BALB/c mice by transplanting BCR-ABL-transduced donor bone marrow cells into lethally irradiated recipient mice. The B-ALL mice were treated with intraperitoneally injected curcumin (40 mg/kg, once a day), and the treated mice survived significantly longer than the vehicle-treated B-ALL mice (p=0.04; Peto-Wilcoxon test). Taken together, our results suggest a potential use of curcumin in treating BCR-ABL-induced B-ALL.

FGX: a frequentist gene expression index for Affymetrix arrays

We consider a new frequentist gene expression index for Affymetrix oligonucleotide DNA arrays, using a similar probe intensity model as suggested by Hein and others (2005), called the Bayesian gene expression index (BGX). According to this model, the perfect match and mismatch values are assumed to be correlated as a result of sharing a common gene expression signal. Rather than a Bayesian approach, we develop a maximum likelihood algorithm for estimating the underlying common signal. In this way, estimation is explicit and much faster than the BGX implementation. The observed Fisher information matrix, rather than a posterior credibility interval, gives an idea of the accuracy of the estimators. We evaluate our method using benchmark spike-in data sets from Affymetrix and GeneLogic by analyzing the relationship between estimated signal and concentration, i.e. true signal, and compare our results with other commonly used methods.

Rapid Discrimination of Fish Pathogenic Vibrio and Photobacterium Species by Oligonucleotide DNA Array

We developed a low-density oligonucleotide DNA array for the discrimination of 17 Vibrio and 1 Photobacterium species that are pathogenic to aquatic animals. We designed oligonucleotide probes targeting non-coding regions of the intergenic transcribed spacers (ITS) between 16S and 23S ribosomal DNA. Based on the alignment of ITS sequences obtained from the database and the results in this study, three oligonucleotide probes from a bacterial species were designed and immobilized on a nylon membrane. The ITS regions were PCR-amplified using a pair of universal primers, followed by hybridization of the digoxigenin (DIG)-labeled PCR products to the membrane. Specific signals were produced with alkaline phosphatase-conjugated anti-DIG antibody and chemiluminescent substrate. Although cross hybridization was observed, the patterns from each species were different among the species. This DNA array is useful for a rapid and easy discrimination of Vibrio species. The total process, including PCR amplification, can be carried out within a day. Our method enables a global screening of fish pathogenic Vibrio and Photobacterium species

Molecular genetic alterations and gene expression profile of a malignant rhabdoid tumor of the kidney

Malignant rhabdoid tumor of the kidney (MRTK) is a rare but highly aggressive tumor in children, and knowledge about the molecular signature of this tumor is limited. We report the molecular genetic alterations and gene expression profile of an MRTK tumor that arose in a 4-month-old Japanese girl. Fluorescence in situ hybridization and Southern blot analyses revealed a homozygous deletion of an ∼0.29-Mb genomic region bordered by the Rgr and DDT genes in these tumor cells. This deleted region encodes SMARCB1, a candidate tumor suppressor gene for MRTK. Using a high-density oligonucleotide DNA array, we found increased expression of 25 genes, including genes involved in the cell cycle (10 genes), DNA replication (3 genes), cell growth (5 genes), and cell proliferation (5 genes), in this MRTK tumor sample, compared with a noncancerous kidney (NK) sample. On the other hand, 64 genes, including 4 genes regulating apoptosis, were found to show decreased expression in this MRTK tumor sample, compared with the NK sample. Among these alterations, we found alterations of expression of some genes, such as IGF2, MDK, TP53, and TNFSF10, in this MRTK tumor, as described previously. The molecular genetic alterations and altered pattern of gene expression found in this case may have contributed to the biological characteristics of the MRTK tumor that arose in our patient.

Oligonucleotide arrays: information from replication and spatial structure

The introduction of oligonucleotide DNA arrays has resulted in much debate concerning appropriate models for the measurement of gene expression. By contrast, little account has been taken of the possibility of identifying the physical imperfections in the raw data. This paper demonstrates that, with the use of replicates and an awareness of the spatial structure, deficiencies in the data can be identified, the possibility of their correction can be ascertained and correction can be effected (by use of local scaling) where possible. The procedures were motivated by data from replicates of Arabidopsis thaliana using the GeneChip ATH1-121501 microarray. Similar problems are illustrated for GeneChip Human Genome U133 arrays and for the newer and larger GeneChip Wheat Genome microarray. R code is freely available on request.