Search PubMed⌕ Search

SEARCH · Search PubMed

Results for “Oligonucleotide Array Sequence Analysis”

Search indexed PubMed citations on genomics, clinical trials, systematic reviews and public health. Explore titles, authors and supplied subject terms, then open the PubMed record.

Quote a phrase for an exact phrase match. Source license links do not imply unrestricted reuse.

At least 883 records · Page 49Linked to original sources
PubMed2004

Combining gene expression data from different generations of oligonucleotide arrays.

BACKGROUND: One of the important challenges in microarray analysis is to take full advantage of previously accumulated data, both from one's own laboratory and from public repositories. Through a comparative analysis on a variety of datasets, a more comprehensive view of the underlying mechanism or structure can be obtained. However, as we discover in this work, continual changes in genomic sequence annotations and probe design criteria make it difficult to compare gene expression data even from different generations of the same microarray platform. RESULTS: We first describe the extent of discordance between the results derived from two generations of Affymetrix oligonucleotide arrays, as revealed in cluster analysis and in identification of differentially expressed genes. We then propose a method for increasing comparability. The dataset we use consists of a set of 14 human muscle biopsy samples from patients with inflammatory myopathies that were hybridized on both HG-U95Av2 and HG-U133A human arrays. We find that the use of the probe set matching table for comparative analysis provided by Affymetrix produces better results than matching by UniGene or LocusLink identifiers but still remains inadequate. Rescaling of expression values for each gene across samples and data filtering by expression values enhance comparability but only for few specific analyses. As a generic method for improving comparability, we select a subset of probes with overlapping sequence segments in the two array types and recalculate expression values based only on the selected probes. We show that this filtering of probes significantly improves the comparability while retaining a sufficient number of probe sets for further analysis. CONCLUSIONS: Compatibility between high-density oligonucleotide arrays is significantly affected by probe-level sequence information. With a careful filtering of the probes based on their sequence overlaps, data from different generations of microarrays can be combined more effectively.

Biopsy↗
PubMed2003

Heterogeneity in control of mRNA stability by AU-rich elements.

AU-rich elements (AREs), located in the 3'-untranslated region of unstable cytokine and chemokine mRNAs, promote rapid decay of otherwise stable mRNAs and may mediate selective mRNA stabilization in response to stimulation with interleukin-1 (IL-1). AREs vary considerably, however, in both size and sequence context. To assess the heterogeneity involved in control of mRNA stability by ARE motifs, human mRNA sequences from IL-1alpha-stimulated HEK293 cells and T98G cells were screened for either instability or stability using both cDNA (950 ARE containing sequences) and Affymetrix oligonucleotide (U95Av2 GeneChip) array analysis. Although ARE-containing mRNAs exhibited a broad range of stability, IL-1alpha promoted stability in a subset of mRNAs that were unstable when transcriptionally induced by tumor necrosis factor alpha. Stabilization of granulocyte/macrophage-colony stimulating factor and IL-8 mRNAs by IL-1alpha was achieved only after 2 h of stimulation, required ongoing protein synthesis, and depended on the activation of p38 MAPK. In contrast, stabilization of Gro3 mRNA in response to IL-1alpha was achieved immediately and was insensitive to inhibitors of protein synthesis and p38 MAPK activation. In concert, these findings demonstrate that ARE sequences are functionally heterogeneous; only a subset of unstable mRNAs is sensitive to stabilization by IL-1alpha. Moreover, IL-1alpha promotes stabilization of unstable mRNAs through distinct mechanistic pathways that distinguish between specific mRNA sequences.

3' Untranslated Regions↗
PubMed2001

Attachment of benzaldehyde-modified oligodeoxynucleotide probes to semicarbazide-coated glass.

Attachment of oligodeoxynucleotides (ODNs) containing benzaldehyde (BAL) groups to semicarbazide-coated glass (SC-glass) slides is described. 5'-BAL-ODNs are prepared using automated DNA synthesis and an acetal-protected BAL phosphoramidite reagent. The hydrophobic protecting group simplifies purification of BAL-ODNs by reverse phase HPLC and is easily removed using standard acid treatment. The electrophilic BAL-ODNs are stable in solution, but react specifically with semicarbazide groups to give semicarbazone bonds. Glass slides were treated with a semicarbazide silane to give SC-glass. BAL-ODNs are coupled to the SC-glass surface by a simple one-step procedure that allows rapid, efficient and stable attachment. Hand-spotted arrays of BAL-ODNs were prepared to evaluate loading density and hybridization properties of immobilized probes. Hybridization to radiolabeled target strands shows that at least 30% of the coupled ODNs were available for hybridization at maximum immobilization density. The array was used to probe single nucleotide polymorphisms in synthetic DNA targets, and PCR products were correctly genotyped using the same macroarray. Application of this chemistry to manufacturing of DNA microarrays for sequence analysis is discussed.

Benzaldehydes↗
PubMed2002

High-density genechip oligonucleotide probe arrays.

High-density DNA probe arrays provide a highly parallel approach to nucleic acid sequence analysis that is transforming gene-based biomedical research. Photolithographic DNA synthesis has enabled the large-scale production of GeneChip probe arrays containing hundreds of thousands of oligonucleotide sequences on a glass "chip" about 1.5 cm2 in size. The manufacturing process integrates solid-phase photochemical oligonucleotide synthesis with lithographic techniques similar to those used in the microelectronics industry. Due to their very high information content, GeneChip probe arrays are finding widespread use in the hybridization-based detection and analysis of mutations and polymorphisms ("genotyping"), and in a wide range of gene expression studies.

Base Sequence↗
PubMed2006

SuperSAGE array: the direct use of 26-base-pair transcript tags in oligonucleotide arrays.

We developed a new platform for genome-wide gene expression analysis in any eukaryotic organism, which we called SuperSAGE array. The SuperSAGE array is a microarray onto which 26-bp oligonucleotides corresponding to SuperSAGE tag sequences are directly synthesized. A SuperSAGE array combines the advantages of the highly quantitative SuperSAGE expression analysis with the high-throughput microarray technology. We demonstrated highly reproducible gene expression profiling by the SuperSAGE array for 1,000 genes (tags) in rice. We also applied this technology to the detailed study of expressed genes identified by SuperSAGE in Nicotiana benthamiana, an organism for which sufficient genome sequence information is not available. We propose that the SuperSAGE array system represents a new paradigm for microarray construction, as no genomic or cDNA sequence data are required for its preparation.

Base Pairing↗
PubMed2000

Identification of differential gene expression by high throughput analysis.

High throughput analysis of differential gene expression is a powerful tool that can be applied to many areas in molecular cell biology, including differentiation, development, physiology, and pharmacology. In recent years, a variety of techniques have been developed to analyze differential gene expression, including comparative expressed sequence tag sequencing, differential display, representational difference analysis, cDNA or oligonucleotide arrays, and serial analysis of gene expression. This review explains the technologies, their scopes, impact on science, as well as their costs and possible limitations. The application of differential display is presented as a tool to identify genes induced by darkness or yellowing process in rice leaves.

Expressed Sequence Tags↗
PubMed2000

Simultaneous detection and genotyping of "Norwalk-like viruses" by oligonucleotide array in a reverse line blot hybridization format.

"Norwalk-like viruses" (NLVs) are the most common cause of outbreaks of nonbacterial gastroenteritis worldwide. To date, the method most widely used for typing of NLV strains is sequencing and subsequent phylogenetic analysis of reverse transcription (RT)-PCR products, which has revealed the existence of stable distinct lineages (genotypes). This typing method is rather costly, not routinely used in clinical laboratories, and not very suitable for the analysis of large numbers of samples. Therefore, we have developed a rapid and simple method for genotyping of NLVs. The method, designated reverse line blot hybridization, is based on the nucleotide divergence of a region of the gene for RNA polymerase which can be used to classify NLVs into genotypes. NLV RNA was amplified by RT-PCR and then hybridized to 18 different membrane-bound oligonucleotides that were able to discriminate among 13 NLV genotypes. Application of the method to a panel of 132 positive stool samples from 34 outbreaks and 20 sporadic cases of gastroenteritis collected in a 6-year period (1994 to 1999) resulted in successful genotyping of 124 samples (94%), as confirmed by phylogenetic analysis. The nucleotide sequences of the remaning eight strains (6%) from three outbreaks did not cluster with the known NLV genotypes. Phylogenetic analysis of the complete and partial open reading frame 2 (capsid gene) sequences of these strains revealed the existence of one novel genotype (Alphatron) and one potentially novel genotype (Amsterdam). This novel method, which allows simultaneous detection and genotyping of NLVs, is useful in the diagnosis and typing of NLVs obtained from outbreaks and in large-scale epidemiological studies.

Caliciviridae Infections↗
PubMed2001

Microfluidic arrays in genetic analysis.

The goal of genetic analysis is to discover genetic markers that are informative for providing high confidence, positive predictive value in managing phenotypic outcomes. Primary consensus sequence data, genetic polymorphism databases and associated phenotype data are rapidly making genetic analysis more useful. Therefore, genetic analysis applications are gradually becoming more mainstream. The diversity and complexity of genetic analysis currently requires an array of analytical techniques, instrument platforms and software to support all the steps from data acquisition to interpretation. As supporting research technologies mature, they are incorporating increasing levels of automation, system integration and miniaturization. Microfluidic arrays are positioned to play a key role in routine genetic analysis, particularly as they begin to appear in more fully integrated analytical platforms.

Animals↗
PubMed2005

Surface plamon resonance imaging of DNA based biosensors for potential applications in food analysis.

The adsorption processes of oligonucleotides immobilised onto suitable photolithographic patterned gold substrates have been investigated in aqueous buffer solution by using a home made surface plasmon resonance (SPR) imaging equipment. A rapid self-assembled method for the construction of DNA chips to be used in SPR imaging experiments have been followed. The immobilised DNA molecules (probes) adopted in our SPR experiments anchored to a gold surface via thiol group were 5'thiol-modified containing a (CH(2))(15) tail. The hybridisation processes taking place with its complementary sequence have been observed and characterized by monitoring phenomena by a SPR imaging system. The two analysed oligonucleotides (probes and target) are of interest in plant gene biotechnological application and differing for the presence at the 5'-end of a poly T16 spacer. Dynamic investigation of smallest changes in SPR imaging pictures performed in liquid phase in the presence of DNA complementary probes have been performed. Quantitative information in terms of threshold of sensitivity has been extracted by using a specific images treatment.

Biosensing Techniques↗
PubMed2006

Childhood acute lymphoblastic leukemia in the age of genomics.

The recent sequencing of the human genome and technical breakthroughs now make it possible to simultaneously determine mRNA expression levels of almost all of the identified genes in the human genome. DNA "chip" or microarray technology holds great promise for the development of more refined, biologically-based classification systems for childhood ALL, as well as the identification of new targets for novel therapy. To date gene expression profiles have been described that correlate with subtypes of ALL defined by morphology, immunophenotype, cytogenetic alterations, and response to therapy. Mechanistic insights into treatment failure have come from the definition of mRNA signatures that predict in vitro chemoresistance, as well as differences between blasts at relapse and new diagnosis. New bioinformatics tools optimize data mining, but validation of findings is essential since "over-fitting" the data is a common danger. In the future, genomic analysis will be complemented by evaluation of the cancer proteome.

Child↗
PubMed2005

A novel optical biosensor format for the detection of clinically relevant TP53 mutations.

The TP53 gene has been the subject of intense research since the realisation that inactivation of this gene is common to most cancer types. Numerous publications have linked TP53 mutations in general or at specific locations to patient prognosis and therapy response. The findings of many studies using general approaches such as immunohistochemistry or sequencing are contradictory. However, the detection of specific mutations, especially those occurring in the structurally important L2 and L3 zinc binding domains, which are the most common sites of TP53 mutations, have been linked to patient prognosis and more strongly to radiotherapy and chemotherapy resistance in several major cancers. In this study, the TI-SPR-1 surface plasmon resonance system and Texas Instruments Spreeta chips were used to develop a DNA biosensor based on thiolated probes complementary to these domains. The sensors were able to detect these mutations in both oligonucleotides and PCR products with normal and mutant TP53 DNA, but the difference in hybridisation signal was small. Preliminary experiments to enhance the signal using Escherichia coli mismatch repair proteins, MutS and single strand binding protein were carried out. It was found that MutS was unable to bind to mismatch oligonucleotides, but single strand binding protein was able to bind to single stranded probes, which had not hybridised to the target, resulting in a three-fold increase in the sensitivity of the biosensor. While further work needs to be carried out to optimise the system, these preliminary experiments indicate that the TI-SPR-1 can be used for the detection of clinically relevant mutations in the TP53 gene and that the sensitivity can be increased significantly using single strand binding protein. This system has a number of advantages over current mutation detection technologies, including lower cost, ease of sensor preparation and measurement procedures, technical simplicity and increased speed due to the lack of need for gel electrophoresis.

Biosensing Techniques↗
PubMed2006

Increased epidermal growth factor receptor gene copy number is associated with poor prognosis in head and neck squamous cell carcinomas.

PURPOSE: High epidermal growth factor receptor (EGFR) gene copy number is associated with poor prognosis in lung cancer, but such findings have not been reported for HNSCC. A better understanding of the EGFR pathway may improve the use of EGFR inhibitors in HNSCC. PATIENTS AND METHODS: EGFR status was analyzed in 86 tumor samples from 82 HNSCC patients by fluorescent in situ hybridization (FISH) to determine EGFR gene copy number, by polymerase chain reaction and direct sequencing for activating mutations, and by DNA microarray and immunohistochemistry for RNA and protein expression. The results were associated with patient characteristics and clinical end points. RESULTS: Forty-three (58%) of 75 samples with FISH results demonstrated EGFR high polysomy and/or gene amplification (FISH positive). The FISH-positive group did not differ from the FISH-negative group with respect to age, sex, race, tumor grade, subsites and stage, or EGFR expression by analyses of RNA or protein. No activating EGFR mutations were found. However, the FISH-positive group was associated with worse progression-free and overall survival (P < .05 and P < .01, respectively; log-rank test). When microarray data were interrogated using the FISH results as a supervising parameter, ECop (which is known to coamplify with EGFR and regulate nuclear factor-kappa B transcriptional activity) had higher expression in FISH-positive tumors. CONCLUSION: High EGFR gene copy number by FISH is frequent in HNSCC and is a poor prognostic indicator. Additional investigation is indicated to determine the biologic significance and implications for EGFR inhibitor therapies in HNSCC.

Aneuploidy↗
PubMed2005

Temporal and parental-specific expression of imprinted genes in a newly derived Chinese human embryonic stem cell line and embryoid bodies.

Although the study of imprinted genes in human development is very important, little is known about their expression and regulation in the early differentiation of human tissues due to lack of an appropriate model. In this study, a Chinese human embryonic stem (hES) cell line, SHhES1, was derived and fully characterized. Expression profiles of human imprinted genes were determined by Affymetrix Oligo micro-array in undifferentiated SHhES1 cells and SHhES1-derived embryoid bodies (EBs) at day 3, 8, 13 and 18. Thirty-two known human imprinted genes were detected in undifferentiated ES cells. Significantly, differential expression was found in nine genes at different stages of EB formation. Expression profile changes were confirmed by quantitative real-time reverse transcriptase-polymerase chain reaction in SHhES1 cells as well as in another independently derived hES cell line, HUES-7. In addition, the monoallelic expressions of four imprinted genes were examined in three different passages of undifferentiated ES cells and EBs of both hES cell lines. The monoallelic expressions of imprinted genes, H19, PEG10, NDNL1 and KCNQ1 were maintained in both undifferentiated hES cells and derived EBs. More importantly, with the availability of maternal peripheral blood lymphocyte sample, we demonstrated that the maternal expression of KCNQ1 and the paternal expression of NDNL1 and PEG10 were maintained in SHhES1 cells. These data provide the first demonstration that the parental-specific expression of imprinted genes is stable in EBs after extensive differentiation, also indicating that in vitro fertilization protocol does not disrupt the parental monoallelic expression of the imprinted genes examined.

Antigens, Neoplasm↗
PubMed2005

Generation of an oligonucleotide array for analysis of gene expression in Chlamydomonas reinhardtii.

The availability of genome sequences makes it possible to develop microarrays that can be used for profiling gene expression over developmental time, as organisms respond to environmental challenges, and for comparison between wild-type and mutant strains under various conditions. The desired characteristics of microarrays (intense signals, hybridization specificity and extensive coverage of the transcriptome) were not fully met by the previous Chlamydomonas reinhardtii microarray: probes derived from cDNA sequences (approximately 300 bp) were prone to some nonspecific cross-hybridization and coverage of the transcriptome was only approximately 20%. The near completion of the C. reinhardtii nuclear genome sequence and the availability of extensive cDNA information have made it feasible to improve upon these aspects. After developing a protocol for selecting a high-quality unigene set representing all known expressed sequences, oligonucleotides were designed and a microarray with approximately 10,000 unique array elements (approximately 70 bp) covering 87% of the known transcriptome was developed. This microarray will enable researchers to generate a global view of gene expression in C. reinhardtii. Furthermore, the detailed description of the protocol for selecting a unigene set and the design of oligonucleotides may be of interest for laboratories interested in developing microarrays for organisms whose genome sequences are not yet completed (but are nearing completion).

Animals↗
PubMed2004

Invasive cleavage reactions on DNA-modified diamond surfaces.

Recently developed DNA-modified diamond surfaces exhibit excellent chemical stability to high-temperature incubations in biological buffers. The stability of these surfaces is substantially greater than that of gold or silicon surfaces, using similar surface attachment chemistry. The DNA molecules attached to the diamond surfaces are accessible to enzymes and can be modified in surface enzymatic reactions. An important application of these surfaces is for surface invasive cleavage reactions, in which target DNA strands added to the solution may result in specific cleavage of surface-bound probe oligonucleotides, permitting analysis of single nucleotide polymorphisms (SNPs). Our previous work demonstrated the feasibility of performing such cleavage reactions on planar gold surfaces using PCR-amplified human genomic DNA as target. The sensitivity of detection in this earlier work was substantially limited by a lack of stability of the gold surface employed. In the present work, detection sensitivity is improved by a factor of approximately 100 (100 amole of DNA target compared with 10 fmole in the earlier work) by replacing the DNA-modified gold surface with a more stable DNA-modified diamond surface.

Apolipoproteins E↗
PubMed2002

Telomerase as a prognostic marker in breast cancer: high-throughput tissue microarray analysis of hTERT and hTR.

Telomerase activity (TA) has been shown to correlate with poor clinical outcome in various tumour entities, indicating that tumours expressing this enzyme may be more aggressive and that TA may be a useful prognostic marker. For breast cancer, however, TA is a controversial prognostic marker; whereas some studies suggest an association between TA and disease outcome, others do not find this association. This study used tissue microarrays (breast carcinoma prognosis arrays) containing 611 samples (each 0.6 mm in diameter) from the tumour centre of paraffin-embedded breast carcinomas to analyse the catalytic subunit of telomerase, human telomerase reverse-transcriptase (hTERT), and the internal RNA component (hTR), which are the core components of the telomerase holoenzyme complex. hTERT protein expression was obtained by immunohistochemistry (human anti-telomerase antibody Ab-2, Calbiochem), and hTR RNA was measured by radioactive in situ hybridization. hTERT and hTR expression were determined semi-quantitatively and graded (scores 1-4). Clinical data, such as histological subtype, pT stage, tumour diameter, pN stage, BRE grade, tumour-specific survival (in months), patient's age and others, were available for statistical analysis. A statistically significant correlation was found between tumour-specific survival (overall survival) and hTERT expression (p < 0.0001) or hTR expression (p = 0.00110). Tumours with higher scores (scores 3, 4) for hTR and/or hTERT were associated with a worse prognosis. In multivariate analysis, hTERT expression was an independent prognostic factor. Previous studies, focusing on analysis of TA in smaller numbers of fresh-frozen breast carcinomas by the TRAP assay, gave controversial results with respect to TA as a prognostic marker. Using tissue microarrays from 611 breast carcinomas, this study has demonstrated that increased expression levels of the telomerase core components, hTERT and hTR, are associated with lower overall survival. These findings suggest that TA should be included in future validation studies as a prognostic marker in breast cancer.

Adult↗
PubMed2003

Loss of CD59 expression in breast tumours correlates with poor survival.

CD59 (protectin), a phosphatidylinositol-anchored glycoprotein, is a member of the cell membrane-bound complement regulatory proteins that inhibits the formation of the terminal membrane attack complex (MAC) of complement. In this study, the expression of CD59 was evaluated in 520 breast carcinomas from patients with a mean follow-up of 87 months. This expression was correlated with clinicopathological features and patient survival. Marked variation in the intensity of CD59 expression, which correlated with histological grade and Nottingham prognostic index (NPI), was found, with higher expression of CD59 found more often in well and moderately differentiated tumours and those of good prognosis (NPI < or = 3.4). In contrast, high grade and poor prognosis (NPI > 5.4) carcinomas significantly demonstrated lack of CD59 expression (p < 0.001). Moreover, it was found that the percentage of CD59-positive cells correlated significantly with patient survival, ie patients with a high percentage of positive cells (>50%) had a better overall survival (p = 0.006). A correlation was also found between the percentage of CD59-positive cells and tumour type and also the development of distant metastases. No association was found between either the intensity or the percentage of cells expressing CD59 and vascular invasion, lymph node stage, tumour size, patient age or menopausal status. In multivariate analysis, CD59 percentage positivity was of independent prognostic significance with grade and lymph node stage. These findings indicate that loss of CD59 may offer a selective advantage for breast cancers, resulting in more aggressive tumours and conferring a poor prognosis for patients.

Adult↗
PubMed2006

Metastasis suppressor gene Raf kinase inhibitor protein (RKIP) is a novel prognostic marker in prostate cancer.

BACKGROUND: Diminished expression of Raf kinase inhibitor protein (RKIP), an inhibitor of the Raf signaling cascade, promotes prostate cancer (PCa) metastasis in a murine model, suggesting that it is a metastasis suppressor gene. However, the prognostic significance of RKIP expression and its association with metastasis in PCa patients is unknown. METHODS: To investigate RKIP protein expression is a prognostic marker in PCa we performed immunohistochemical staining for RKIP expression in tissue microarrays consisting of 758 non-neoplastic prostate tissues, primary tumors and metastases from 134 PCa patients. The Cox proportional-hazards model was used to adjust for covariates including Gleason score, tumor volume, tumor weight, clinical stage, digital rectal exam findings, serum PSA level and surgical margins. RESULTS: RKIP expression was low in approximately 5%, 48%, and 89% of non-neoplastic prostate, primary tumors and metastases, respectively. Low RKIP expression in primary tumors was a strong positive predictive factor for PCa recurrence based on PSA levels. In patients whose primary tumors expressed high RKIP levels, the 7-year PSA recurrence rate was <10%; whereas in patients with tumors with low RKIP expression the recurrence rate was 50% (P<0.001). Multivariate analysis revealed RKIP was an independent prognostic factor (P<0.001). CONCLUSION: In contrast to increased expression of pro-tumorigenic genes, these results demonstrate decreased protein expression of a gene, for example, RKIP, can serve as a prognostic marker in PCa patients.

Biomarkers, Tumor↗