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Results for “Oligonucleotide Array Sequence Analysis”

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PubMed2006

Semiquantitative in situ hybridization using radioactive probes to study gene expression in motoneuron populations.

In situ hybridization is a powerful technique for examining changes in mRNA levels. Its primary advantage over the Northern blot and reverse transcription polymerase chain reaction is the ability to localize specific species of mRNA to a particular cell population in a heterocellular system. This consideration is important when studying gene expression, especially in areas of high cellular heterogeneity, such as the central nervous system, where the differentiation between neuronal and glial gene expression is critical in evaluating the effects of physiological or pathological stimuli. We have used radioactive in situ hybridization to study changes of mRNA levels in several motoneuron populations following axonal injury. Considering that many neuronal populations have multiple targets of innervation, we used in situ hybridization in a manner that focused on the examination of changes in gene expression at the single cell level. This chapter describes the in situ hybridization protocol that our laboratory has used to demonstrate alterations in gene expression in specific motoneurons whose cell bodies are localized to the central nervous system.

Animals↗
PubMed2005

Diffusion, mixing, and associated dye effects in DNA-microarray hybridizations.

Typical DNA microarrays utilize diffusion of dye-labeled cDNA probes followed by sequence-specific hybridization to immobilized targets. Here we experimentally estimated the distance typical probes travel during static 16-h hybridizations. Probes labeled with Cy3 and Cy5 were individually introduced to opposite sides of a microarray with minimal convective mixing. Oppositely labeled probes diffused across the initial front separating the two solutions, generating a zone with both dyes present. Diffusion-distance estimates for Cy3- and Cy5-labeled cDNAs were 3.8 mm and 2.6 mm, respectively, despite having almost identical molecular masses. In separate 16-h hybridization experiments with oppositely labeled probes premixed, arrays that were continuously mixed had 15-20% higher signal intensities than arrays hybridized statically. However, no change was observed in the Cy3/Cy5 signal intensity ratio between continuously mixed and static hybridizations. This suggests that the observed dye bias in diffusion-distance estimates results from differences in the detection limits of Cy3 and Cy5-labeled cDNA, a potential concern for array data on low-abundance transcripts. Our conservative diffusion-distance estimates indicate that replicate targets >7.6 mm apart will not compete for scarce probes. Also, raising the microarray gap height would delay the onset of diffusion-limited hybridization by increasing the amount of available probe.

Algorithms↗
PubMed2004

Statistical framework for phylogenomic analysis of gene family expression profiles.

Microarray technology has produced massive expression data that are invaluable for investigating the genome-wide evolutionary pattern of gene expression. To this end, phylogenetic expression analysis is highly desirable. On the basis of the Brownian process, we developed a statistical framework (called the E(0) model), assuming the independent expression of evolution between lineages. Several evolutionary mechanisms are integrated to characterize the pattern of expression diversity after gene duplications, including gradual drift and dramatic shift (punctuated equilibrium). When the phylogeny of a gene family is given, we show that the likelihood function follows a multivariate normal distribution; the variance-covariance matrix is determined by the phylogenetic topology and evolutionary parameters. Maximum-likelihood methods for multiple microarray experiments are developed, and likelihood-ratio tests are designed for testing the evolutionary pattern of gene expression. To reconstruct the evolutionary trace of expression diversity after gene (or genome) duplications, we developed a Bayesian-based method and use the posterior mean as predictors. Potential applications in evolutionary genomics are discussed.

Algorithms↗
PubMed2005

Altered patterns of gene expression specific to thoracic aortic aneurysms: microarray analysis of surgically resected specimens.

Changes in the expression levels of several genes have been described in aortic aneurysm specimens, however, the spectrum of diverse molecular alterations remains to be elucidated. We attempted to identify key molecules that modulate the pathogenesis of aortic aneurysm, using a complimentary DNA microarray carrying approximately 13,000 human genes. Segments of thoracic aortic aneurysms (TAA) and adjacent normal thoracic aortic tissues without aneurysmal changes (NTA) were obtained from 20 patients undergoing graft surgery. RNA obtained from five pairs of TAA and NTA samples was compared to determine aneurysm-specific alterations using microarray. Further, the expression levels of several genes of interest were verified in the remaining specimens by real-time reverse transcription-polymerase chain reaction (RT-PCR). In microarray assays, several types of the matrix metalloproteinases were upregulated as reported previously. Also, 220 genes suggested to be involved in protein degradation, inflammation, apoptosis, stress response, intracellular signaling, and other processes were significantly upregulated. Many of these genes have not been previously implicated in cardiovascular disease. The real time RT-PCR independently confirmed that the expression levels of MMP-2, MMP-9, ADAMTS-1, and caspase 4 were consistently increased in TAA. The results indicate that many genes are involved in a complicated manner in the pathogenesis of TAA. Investigation of these genes will help clarify the pathogenesis of this disease, and may lead to the discovery of novel therapeutic targets.

ADAM Proteins↗
PubMed2003

Single nucleotide polymorphism genotyping using locked nucleic acid (LNA).

Locked nucleic acid (LNA) is a new class of bicyclic high affinity DNA analogs. LNA-containing oligonucleotides confer significantly increased affinity against their complementary DNA targets, increased mismatch discrimination (delta Tm) and allow full control of the melting point of the hybridization reaction. LNA chemistry is completely compatible with the traditional DNA phosphoramidite chemistry and therefore LNA-DNA mixmer oligonucleotides can be designed with complete freedom for optimal performance. These properties render LNA oligonucleotides very well suited for SNP genotyping and have enabled several approaches for enzyme-independent SNP genotyping based on allele-specific hybridization. In addition, allele-specific PCR assays relying on enzymatically-enhanced discrimination can be improved using LNA-modified oligonucleotides. The use of LNA transforms enzyme-independent genotyping approaches into experimentally simple, robust and cost-effective assays, which are highly suited for genotyping in clinical and industrial settings.

Alleles↗
PubMed2005

Altered expression of TFF-1 and CES-2 in Barrett's Esophagus and associated adenocarcinomas.

Identification of biomarkers to recognize individuals with Barrett's esophagus (BE) predisposed to develop malignancy is currently a pressing issue. We utilized gene expression profiling to compare molecular signatures of normal esophagus and stomach, BE, and adenocarcinoma (AC) to identify such potential biomarkers. Over 22,000 genes were analyzed by oligonucleotide microarrays on 38 unique RNA Unsupervised and supervised clusterings were performed on a subset of 2849 genes that varied most significantly across the specimens. Immunohistochemistry (IHC) for two of the significantly differentially expressed gene products was performed on tissue microarrays. Unsupervised clustering identified two discernable molecular BE profiles, one of which was similar to normal gastric tissue ("BE1"), and another that was shared by several of the AC specimens ("BE2"). The BE1 profile included expression of several genes that have been described as tumor-suppressor genes, most notably trefoil factor 1 (TFF-1). The BE2 profile included expression of genes previously found overexpressed in cancers, such as carboxylesterase-2 (CES-2). IHC demonstrated the loss of TFF-1 late in the progression of BE to AC. It also revealed CES-2 as being upregulated in AC documented to have arisen in the presence of BE. These potential biomarkers, as well as the relative expression of genes from BE1 versus those from BE2, may be validated in the future to aid in risk stratification and guide treatment protocols in patients with BE and associated AC.

Adenocarcinoma↗
PubMed2006

Constitutive activation of neuregulin/ERBB3 signaling pathway in clear cell sarcoma of soft tissue.

Clear cell sarcoma of soft tissue (CCSST) represents a highly malignant tumor of the musculoskeletal system that is characterized by the chromosomal translocation t(12;22)(q13;q12) of the Ewing sarcoma gene (EWSR1) and activating transcription factor 1 (ATF1). In a former microarray expression study, we identified ERBB3, a member of the epidermal growth factor receptor (EGFR) family, as a promising new diagnostic marker in the differential diagnosis of CCSST. Here we show that, besides ErbB3, all CCSST cell lines (n = 8) also express the ErbB2 receptor or the ErbB4 receptor, representing an adequate coreceptor of ErbB3. The phosphorylation status of ErbB3 revealed these receptor pairs to be either constitutively activated in CCSST cells with high neuregulin-1 (NRG1) expression (n = 4) or activatable by exogenic NRG1 in cells showing low amounts of NRG1 mRNA (n = 4). Exogenous NRG1 stimulated the growth of a subset of CCSST cells but did not affect the kinetics of another subset. This difference was not strictly dependent on endogenous NRG1 expression; however, the growth-inhibiting effect of the pan-ErbB tyrosine kinase inhibitor CI-1033 or PD158780 clearly correlated with NRG1 expression indicating an autocrine growth stimulation loop which may constitute an interesting target of new therapeutic strategies in this tumor entity.

Alleles↗
PubMed2001

Effector genes altered in MCF-7 human breast cancer cells after exposure to fractionated ionizing radiation.

Understanding the molecular mechanisms involved in the response of tumors to fractionated exposures to ionizing radiation is important for improving radiotherapy and/or radiochemotherapy. In the present study, we examined the expression of stress-related genes in an MCF-7 cell population (MCF-IR20) that has been derived through treatment with fractionated irradiation (2 Gy per fraction with a total dose of 40 Gy). MCF-IR20 cells showed a 1.6-fold increase in sensitization with dose at 10% isosurvival in a clonogenic assay, and a reduced growth delay ( approximately 15 h compared to approximately 27 h), compared to the parental MCF-7 cells treated with a single dose of 5 Gy. To determine which effector genes were altered in the MCF-IR20 cells, the expression of stress-related effector genes was measured using a filter with 588 genes (Clontech) that included major elements involved in cell cycle control, DNA repair, and apoptosis. Compared to MCF-7 cells that were not exposed to fractionated radiation, 19 genes were up- regulated (2.2-5.1-fold) and 4 were down-regulated (2.7-3.4- fold) in the MCF-IR20 cells. In agreement with the array results, 6 up-regulated genes tested by RT-PCR showed elevated expression. Also, activities of the stress-related transcription factors NFKB, TP53 and AP1 showed a 1.2-4.5-fold increase after a single dose of 5 Gy in MCF-IR20 cells compared with parental MCF-7 cells. However, when the radioresistant MCF-IR20 cell were cultured for more than 12 passages after fractionated irradiation (MCF-RV), radioresistance was lost, with the radiosensitivity being the same as the parental MCF- 7 cells. Interestingly, expression levels of CCNB1, CD9 and CDKN1A in MCF-RV cells returned to levels expressed by the parental cells, whereas the expression levels of three other genes, MSH2, MSH6 and RPA remained elevated. To determine if any of the changes in gene expression could be responsible for the induced radioresistance, CCNB1 and CDKN1A, both of which were up-regulated in MCF-IR20 cells and down-regulated in MCF-RV cells, were studied further by transfection with antisense oligonucleotides. Antisense of CCNB1 significantly reduced the clonogenic survival of MCF- IR20 cells at doses of 5 and 10 Gy, from 42% to 26% and from 5.7% to 1.0%, respectively. Antisense of CDKN1A, however, had no effect on radiation survival of MCF-IR20 cells. In summary, these results suggest that stress-related effector genes are altered in cells after treatment with fractionated irradiation, and that up-regulation of CCNB1 is responsible, at least in part, for radioresistance after fractionated irradiation.

Adenocarcinoma↗
PubMed2006

Reliable and fast allele-specific extension of 3'-LNA modified oligonucleotides covalently immobilized on a plastic base, combined with biotin-dUTP mediated optical detection.

In the present work, a convenient microarray SNP typing system has been developed using a plastic base that covalently immobilizes amino-modified oligonucleotides. Reliable SNP allele discrimination was achieved by using allelic specificity-enhanced enzymatic extension of immobilized oligonucleotide primer, with a locked nucleic acid (LNA) modification at the SNP-discriminating 3'-end nucleotide. Incorporation of multiple biotin-dUTP molecules during primer extension, followed by binding of alkaline phosphatase-conjugated streptavidin, allowed optical detection of the genotyping results through precipitation of colored alkaline phosphatase substrates onto the surface of the plastic base. Notably, rapid primer extension was demonstrated without a preliminary annealing step of double-stranded template DNA, allowing overall processes to be performed within a couple of hours. Simultaneous evaluation of three SNPs in the genes TGFB1, SOD2 and APEX1, previously investigated for association with radiation sensitivity, in 25 individuals has shown perfect assignment with data obtained by another established technique (MassARRAY system).

Alkaline Phosphatase↗
PubMed2006

Fiber optic array biosensors.

Optical fiber arrays provide a powerful substrate for creating high-density sensing systems that can address a variety of biological problems. The fiber substrate can be used to create femtoliter wells that can be loaded with individual beads to create high-density arrays for multiplexed screening and analysis. In addition, living cells can be loaded into the arrays, and their individual responses monitored over long time periods, enabling functional screening of biologically active compounds. Adherent cells can be attached to the fiber substrate to provide a rapid method for observing cell migration and for screening anti-migratory compounds. Finally, individual enzyme molecules can be loaded into the array wells enabling single molecule detection via enzyme-catalyzed signal amplification.

Animals↗
PubMed2001

Fabrication of DNA microarrays using unmodified oligonucleotide probes.

Microarrays printed on glass slides are often constructed by covalently linking oligonucleotide probes to a derivatized surface. These procedures typically require relatively expensive amine- or thiol-modified oligonucleotide probes that add considerable expense to larger arrays. We describe a system by which unmodified oligonucleotide probes are bound to either nonderivatized or epoxy-silane-derivatized glass slides. Biotinylated PCR products are heat denatured, hybridized to the arrays, and detected using an enzymatic amplification system. Unmodified probes appear to detach from the slide surface at high pH (> 10.0), suggesting that hydrogen bonding plays a significant role in probe attachment. Regardless of surface preparation, high temperature (up to 65 degrees C) and low ionic strength (deionized water) do not disturb probe attachment; hence, the fabrication method described here is suitable for a wide range of hybridization stringencies and conditions. We illustrate kinetics of room temperature hybridizations for probes attached to nonderivatized slides, and we demonstrate that unmodified probes produce hybridization signals equal to amine-modified, covalently bound probes. Our method provides a cost-effective alternative to conventional attachment strategies that is particularly suitable for genotyping PCR products with nucleic acid microarrays.

Nucleic Acid Hybridization↗
PubMed2001

Automating parallel peptide synthesis for the production of PNA library arrays.

A system was establishedfor the parallel synthesis of peptide library arrays in afully automated manner Synthesis takes place in blocks made of polyoxymethylene that hold during all synthesis steps a polypropylene membrane of 8 x 12 cm. Yields are in the nanomole range, obtained at a low consumption of reagents. The current setup is based on a commercially available pipetting robot and supports the generation of 1536 different oligomers/run. Much higher array densities are possible because the membranes are amicable to spot diameters of down to 200 microm, naturally at a cost of the absolute amount produced of each oligomer The method was put to use for the creation of arrayed libraries of peptide nucleic acids (PNAs). These can be employed both as a source of PNA molecules applied individually in experimentation subsequent to their release or as intact oligomer arrays in hybridization analyses.

Base Sequence↗
PubMed2003

Microarray-based method for combinatorial library sequence mapping and characterization.

Here we describe a DNA-chip-based method for high-throughput sequence mapping. This involves competitive hybridization between short and differentially labeled fluorescent oligonucleotide probes and glass-supported PCR products. Competition between an excess of oligonucleotide probes targeting the same sequence segment improves sequence discrimination and reduces sensitivity to experimental conditions such as probe concentrations, hybridization, and washing temperatures and durations. The method was found to be particularly adapted to sequence mapping of combinatorial libraries obtained by DNA shuffling between members of a gene family. We present an application of this technique for the characterization of recombination biases in combinatorial libraries used in directed evolution.

Base Sequence↗
PubMed2003

Scoring insertion-deletion polymorphisms by dynamic allele-specific hybridization.

Genome variation provides researchers with thousands of markers with which to study human demographic history and phenotypes. Insertion-deletion (indel) polymorphism is an important and abundant form of human genome variation, and convenient methods for genotyping indels are therefore needed. Here we evaluate dynamic allele-specific hybridization (DASH) for its ability to score indels. Evaluation of six model indel DASH assays based on synthetic oligonucleotides showed that length differences of 1-5 bp were accurately scored. Only single probes were required to assay indels of 3-4 bp or less, while longer indels tended to require the use of both allele probes serially. The best results were obtained by central placing of the probe over the indel. Model study findings were confirmed by running indel DASH assays upon PCR-amplified targets representing four polymorphisms from Alzheimer's disease candidate genes APBB1 and LRP1. These indels were genotyped in a set of 121 patients and 156 controls. While no disease association was found, the data quality confirmed that DASH is a robust and useful procedure for genotyping indels of the size range typically found in the human genome.

Alleles↗
PubMed2004

Evaluation of 50-mer oligonucleotide arrays for detecting microbial populations in environmental samples.

Microarrays fabricated with oligonucleotides longer than 40 bp have been introduced for monitoring whole genome expression but have not been evaluated with environmental samples. To determine the potential of this type of microarray for environmental studies, a 50-mer oligonucleotide microarray was constructed using 763 genes involved in nitrogen cycling: nitrite reductase (nirS and nirK), ammonia monooxygenase (amoA), nitrogenase (nifH), methane monooxygenase (pmoA), and sulfite reductase (dsrAB) from public databases and our own sequence collections. The comparison of the sequences from pure cultures indicated that the developed microarrays could provide species-level resolution for analyzing microorganisms involved in nitrification, denitrification, nitrogen fixation, methane oxidation, and sulfite reduction. Sensitivity tests suggested that the 50-mer oligonucleotide arrays could detect dominant populations in the environments, although sensitivity still needs to be improved. A significant quantitative relationship was also obtained with a mixture of DNAs from eight different bacteria. These results suggest that the 50-mer oligonucleotide array can be used as a specific and quantitative parallel tool for the detection of microbial populations in environmental samples.

Base Sequence↗
PubMed2005

High reproducibility using sodium hydroxide-stripped long oligonucleotide DNA microarrays.

Recently, long oligonucleotide (60- to 70-mer) microarrays for two-color experiments have been developed and are gaining widespread use. In addition, when there is limited availability of mRNA from tissue sources, RNA amplification can and is being used to produce sufficient quantities of cRNA for microarray hybridization. Taking advantage of the selective degradation of RNA under alkaline conditions, we have developed a method to "strip" glass-based oligonucleotide microarrays that use fluorescent RNA in the hybridization, while leaving the DNA oligonucleotide probes intact and usable for a second experiment. Replicate microarray experiments conducted using stripped arrays showed high reproducibility, however, we found that arrays could only be stripped and reused once without compromising data quality. The intraclass correlation (ICC) between a virgin array and a stripped array hybridized with the same sample showed a range of 0.90-0.98, which is comparable to the ICC of two virgin arrays hybridized with the same sample. Using this method, once-stripped oligonucleotide microarrays are usable, reliable, and help to reduce costs.

Breast Neoplasms↗
PubMed2005

Taking arrays from the lab to the field: trying to make sense of the unknown.

With the rapid pace of nucleic acid microarray technology development and a renewed national emphasis on detecting and characterizing microorganisms in environmental samples, there is a rush to operationalize existing microarray technologies and apply them to uncharacterized environmental backgrounds. The purpose of this article is to pause and ask a basic question: what do microarray data actually mean in the face of uncharacterized sample backgrounds? In attempting to answer this question, we draw a clear distinction between hypothesis-driven fundamental science and operational uses of microarray technology; assess microarray technology assumptions in the face of uncharacterized environments; offer an environmental microbiologist's perspective on technology needs and requirements for quantitatively analyzing microbial communities; and hopefully stimulate a scientific and technical dialogue around the concept of analytical environmental microbiology and future technology development.

Bacteria↗