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PubMedDate not supplied

ChipCheck--a program predicting total hybridization equilibria for DNA binding to small oligonucleotide microarrays.

Presented here is the program ChipCheck that allows the computation of total hybridization equilibria for hybridization experiments involving small oligonucleotide arrays. The calculation requires the free energies of binding for all pairs of probes and targets as well as total strand concentrations and probe molecule numbers. ChipCheck has been tested computationally on microarrays with up to 100 spots and 42 target strands (4200 binding equilibria). It arrives at solutions through iterations employing the multidimensional Newton method. While currently running in simulation mode only, an extension of the approach to the exhaustive analysis of chip results is being outlined and may be implemented in the future. The output displays the extent of correct and cross hybridization both graphically and numerically. In principle, calculating total hybridization equilibria allows for eliminating noise from DNA chip results and thus an improvement in sensitivity and accuracy.

Algorithms↗
PubMed2006

Pathway and gene-set activation measurement from mRNA expression data: the tissue distribution of human pathways.

BACKGROUND: Interpretation of lists of genes or proteins with altered expression is a critical and time-consuming part of microarray and proteomics research, but relatively little attention has been paid to methods for extracting biological meaning from these output lists. One powerful approach is to examine the expression of predefined biological pathways and gene sets, such as metabolic and signaling pathways and macromolecular complexes. Although many methods for measuring pathway expression have been proposed, a systematic analysis of the performance of multiple methods over multiple independent data sets has not previously been reported. RESULTS: Five different measures of pathway expression were compared in an analysis of nine publicly available mRNA expression data sets. The relative sensitivity of the metrics varied greatly across data sets, and the biological pathways identified for each data set are also dependent on the choice of pathway activation metric. In addition, we show that removing incoherent pathways prior to analysis improves specificity. Finally, we create and analyze a public map of pathway expression in human tissues by gene-set analysis of a large compendium of human expression data. CONCLUSION: We show that both the detection sensitivity and identity of pathways significantly perturbed in a microarray experiment are highly dependent on the analysis methods used and how incoherent pathways are treated. Analysts should thus consider using multiple approaches to test the robustness of their biological interpretations. We also provide a comprehensive picture of the tissue distribution of human gene pathways and a useful public archive of human pathway expression data.

Cell Line↗
PubMed2006

Single-nucleotide polymorphism genotyping by nanoparticle-enhanced surface plasmon resonance imaging measurements of surface ligation reactions.

A sensitive method for the analysis of single nucleotide polymorphisms (SNPs) in genomic DNA that utilizes nanoparticle-enhanced surface plasmon resonance imaging (SPRI) measurements of surface enzymatic ligation reactions on DNA microarrays is demonstrated. SNP identification was achieved by using sequence-specific surface reactions of the enzyme Taq DNA ligase, and the presence of ligation products on the DNA microarray elements was detected using SPRI through the hybridization adsorption of complementary oligonucleotides attached to gold nanoparticles. The use of gold nanoparticles increases the sensitivity of the SPRI so that single bases in oligonucleotides can be successfully identified at a concentration of 1 pM. This sensitivity is amply sufficient for performing multiplexed SNP genotyping by using multiple PCR amplicons and should also allow for the direct detection and identification of SNP sequences from 1 pM unamplified genomic DNA samples with this array-based and label-free SPRI methodology. As a first example of SNP genotyping, three different human genomic DNA samples were screened for a possible point mutation in the BRCA1 gene that is associated with breast cancer.

DNA↗
PubMed2005

In vitro identification and in silico utilization of interspecies sequence similarities using GeneChip technology.

BACKGROUND: Genomic approaches in large animal models (canine, ovine etc) are challenging due to insufficient genomic information for these species and the lack of availability of corresponding microarray platforms. To address this problem, we speculated that conserved interspecies genetic sequences can be experimentally detected by cross-species hybridization. The Affymetrix platform probe redundancy offers flexibility in selecting individual probes with high sequence similarities between related species for gene expression analysis. RESULTS: Gene expression profiles of 40 canine samples were generated using the human HG-U133A GeneChip (U133A). Due to interspecies genetic differences, only 14 +/- 2% of canine transcripts were detected by U133A probe sets whereas profiling of 40 human samples detected 49 +/- 6% of human transcripts. However, when these probe sets were deconstructed into individual probes and examined performance of each probe, we found that 47% of human probes were able to find their targets in canine tissues and generate a detectable hybridization signal. Therefore, we restricted gene expression analysis to these probes and observed the 60% increase in the number of identified canine transcripts. These results were validated by comparison of transcripts identified by our restricted analysis of cross-species hybridization with transcripts identified by hybridization of total lung canine mRNA to new Affymetrix Canine GeneChip. CONCLUSION: The experimental identification and restriction of gene expression analysis to probes with detectable hybridization signal drastically increases transcript detection of canine-human hybridization suggesting the possibility of broad utilization of cross-hybridizations of related species using GeneChip technology.

Animals↗
PubMed2001

Detection of rifampicin-resistant Mycobacterium tuberculosis strains by hybridization and polymerase chain reaction on a specialized TB-microchip.

Two alternative methods for identification of rifampicin-resistant strains of Mycobacterium tuberculosis on biological microchips are developed. The methods are based on detection of point mutations and other rearrangements in the rpoB gene region determining rifampicin resistance. Hybridization on TB-microchip detects 30 mutant variants of DNA in rifampicin-resistant strains (about 95% of all resistant forms). Allele-specific microchip PCR shortens the duration of analysis to 1.5 h. These methods can be used in clinical diagnostic laboratories for evaluating drug resistance/sensitivity of tuberculosis agent and for monitoring of the efficiency of antibiotic therapy.

Antibiotics, Antitubercular↗
PubMed2005

Monitoring synaptogenesis in the developing mouse cerebellum with an original oligonucleotide microarray.

Use of DNA microarrays in neuroscience have been limited to rough screening purposes, mainly because the reliability and sensitivity of available arrays are not high enough. Because only a few hundred to one thousand genes are usually found to change expression levels in most experiments, we attempted to develop a more quantitative array by the following strategies: 1) limit the genes to those relevant to the experimental system, 2) design oligonucleotide probes of specified molecular properties so that more stringent hybridization conditions can be employed, 3) place six spots per probe on one slide and use multiple normalization genes, and 4) use a new type of gold-coated slide with higher S/N ratio. Genes involved in the construction and functioning of the synapse were selected from the literature as well as experimentally by comparing cerebella from hypothyroid and control mice at postnatal day 15 (P15). Although the number of genes covered was not yet large (172 genes), the custom array "Synaptoarray" thus constructed was capable of detecting +/-20% difference in gene expression ratios. Analysis of the postnatal development of the mouse cerebellum by using Synaptoarray demonstrated a general expression pattern with a peak at P7, followed by a decline at P15 and a partial recovery after P21. P10 clearly marked the end of the initial growth stage and a major transcriptional turning point in this system. This result suggests that such a custom array should be useful in monitoring perturbations to the normal developmental program.

Animals↗
PubMed2002

Analysis of the heat shock response of Neisseria meningitidis with cDNA- and oligonucleotide-based DNA microarrays.

Oligonucleotide- and cDNA-based microarrays comprising a subset of Neisseria meningitidis genes were assessed for study of the meningococcal heat shock response and found to be highly suitable for transcriptional profiling of N. meningitidis. Employing oligonucleotide arrays encompassing the entire genome of N. meningitidis, we analyzed the meningococcal heat shock response on a global scale and identified 55 heat shock-deregulated open reading frames (34 induced and 21 repressed).

DNA, Bacterial↗
PubMed2004

Novel RNAs identified from an in-depth analysis of the transcriptome of human chromosomes 21 and 22.

In this report, we have achieved a richer view of the transcriptome for Chromosomes 21 and 22 by using high-density oligonucleotide arrays on cytosolic poly(A)(+) RNA. Conservatively, only 31.4% of the observed transcribed nucleotides correspond to well-annotated genes, whereas an additional 4.8% and 14.7% correspond to mRNAs and ESTs, respectively. Approximately 85% of the known exons were detected, and up to 21% of known genes have only a single isoform based on exon-skipping alternative expression. Overall, the expression of the well-characterized exons falls predominately into two categories, uniquely or ubiquitously expressed with an identifiable proportion of antisense transcripts. The remaining observed transcription (49.0%) was outside of any known annotation. These novel transcripts appear to be more cell-line-specific and have lower and less variation in expression than the well-characterized genes. Novel transcripts were further characterized based on their distance to annotations, transcript size, coding capacity, and identification as antisense to intronic sequences. By RT-PCR, 126 novel transcripts were independently verified, resulting in a 65% verification rate. These observations strongly support the argument for a re-evaluation of the total number of human genes and an alternative term for "gene" to encompass these growing, novel classes of RNA transcripts in the human genome.

Cell Line↗
PubMed2006

Application of oligonucleotide arrays to high-content genetic analysis.

The scope of single nucleotide polymorphism genotyping for genetic association studies has expanded recently from the use of relatively small numbers of candidate genes and markers, to include hypothesis-free, whole-genome approaches using hundreds of thousands of polymorphisms. The ability to perform such large-scale association studies has been dependant on the development of highly parallel and cost-effective genotyping platforms, of which those based on oligonucleotide arrays have proved to be the most scalable and widely adopted. It is to be expected that the new array-based genotyping methods will not only greatly expand the scope of genetic studies, but, as further content is added to arrays, will also form part of an integrated set of DNA, RNA and proteomic analyses enabling the detailed, multilayered study of complex disease-linked phenotypes.

Base Sequence↗
PubMed2004

Innovative instrumentation for microarray scanning and analysis: application for characterization of oligonucleotide duplexes behavior.

Accuracy in microarray technology requires new approaches to microarray reader development. A microarray reader system (optical scanning array or OSA reader) based on automated microscopy with large field of view, high speed 3 axis scanning at multiple narrow-band spectra of excitation light has been developed. It allows fast capture of high-resolution, multi-fluorescence images and is characterized by a linear dynamic range and sensitivity comparable to commonly used photo-multiplier tube (PMT)-based laser scanner. Controlled by high performance software, the instrument can be used for scanning and quantitative analysis of any type of dry microarray. Studies implying temperature-controlled hybridization chamber containing a microarray can also be performed. This enables the registration of kinetics and melting curves. This feature is required in a wide range of on-chip chemical and enzymatic reactions including on-chip PCR amplification. We used the OSA reader for the characterization of hybridization and melting behaviour of oligonucleotide:oligonucleotide duplexes on three-dimensional Code Link slides.

Image Processing, Computer-Assisted↗
PubMed2004

Comparison of different probe-level analysis techniques for oligonucleotide microarrays.

Three different software packages for the probe-level analysis of high-density oligonucleotide microarray data were compared using an experiment-derived data set that was validated using real-time PCR. The efficiency with which these three programs could identify true positives in this data set was assessed. In addition, estimates of false-positive and false-negative rates were determined. The performance of the programs using very small data sets was also compared, and recommendations for use are suggested.

Animals↗
PubMed2003

Identification and validation of genes involved in the pathogenesis of colorectal cancer using cDNA microarrays and RNA interference.

PURPOSE: The purpose of this study was to profile gene expression changes in colorectal tumors to identify new targets and strategies for the management of this disease. EXPERIMENTAL DESIGN: cDNA microarray analysis was used to detect differences in gene expression between normal tissue and colon tumors and polyps isolated from 20 patients. To identify genes that are important in regulating the growth properties of colorectal cancer, RNA interference (RNAi) was used to disrupt expression of several of the overexpressed genes in a colon tumor cell line, HCT116, which showed similar patterns of gene expression as many of the patient tumors. RESULTS: Expression changes of > or =2-fold in approximately one-third of the patients were consistently observed for 2632 of a total of 9592 genes (574 up-regulated genes and 2058 down-regulated genes). Subsequent analysis of 13 genes by quantitative real-time PCR confirmed the reliability of this analysis. RNAi-mediated disruption of the expression of one of these genes, survivin, a potent inhibitor of apoptosis, severely reduced tumor growth both in vitro and in an in vivo xenograft model. CONCLUSIONS: The combined use of microarray analysis and RNAi provides an excellent system to define the role of specific genes that are up-regulated in cancer lead to the increased in vitro and in vivo growth of colon tumors.

Animals↗
PubMed2003

[Bioinformatic analysis of dengue virus cDNAs and design of oligonucleotide probes for microarray detection of the virus].

OBJECTIVE: To design oligonucleotide probes for microarray detection of dengue virus. METHODS: By analyzing the cDNAs of dengue viruses of 4 different serotypes with BLAST program, a group of specific sequences for the candidate probes was acquired. Oligo6.0 software was applied to analyze the candidates to select the probes with high specificity, identical length and similar melting temperature (Tm). RESULT: Altogether 48 oligonucleotide probes were designed, and deposited on oligonucleotide chips as the microarray for dengue virus detection. CONCLUSION: BLAST program and Oligo6.0 software are simple and effective means for designing the oligonucleotide probes.

Base Sequence↗
PubMed2006

Utilising the left-helical conformation of L-DNA for analysing different marker types on a single universal microarray platform.

L-DNA is the perfect mirror-image form of the naturally occurring d-conformation of DNA. Therefore, L-DNA duplexes have the same physical characteristics in terms of solubility, duplex stability and selectivity as D-DNA but form a left-helical double-helix. Because of its chiral difference, L-DNA does not bind to its naturally occurring D-DNA counterpart, however. We analysed some of the properties that are typical for L-DNA. For all the differences, L-DNA is chemically compatible with the D-form of DNA, so that chimeric molecules can be synthesized. We take advantage of the characteristics of L-DNA toward the establishment of a universal microarray that permits the analysis of different kinds of molecular diagnostic information in a single experiment on a single platform, in various combinations. Typical results for the measurement of transcript level variations, genotypic differences and DNA-protein interactions are presented. However, on the basis of the characteristic features of L-DNA, also other applications of this molecule type are discussed.

Animals↗
PubMed2004

Assessment of 35mer amino-modified oligonucleotide based microarray with bacterial samples.

Parallel quantification of a large number of messenger RNA transcripts, using microarray technology, promises to provide unsuspected information about many cellular processes. Although experimental protocols on microarray applications are available, only limited methodological information on glass-slide manufacturing and signal interpretation has been published. The aim of this paper is to provide new insights into the practical aspects of the construction and hybridization of oligonucleotide-based microarrays. The intracellular symbiotic bacterium of aphids, Buchnera aphidicola, is used here as a model organism. The first part of the work is devoted to the optimization of procedures for printing slides, labeling of cDNA targets and hybridization. In the second part, based on a statistical analysis of the results, we discuss the influence of the probe attachment chemistry, of the labeling method, of the oligonucleotide position and of the concentration of a spotted oligonucleotide on signal intensity. The problem of signal specificity is also addressed, based on the calculation of the fluorescent ratio for each probe to its corresponding mismatch control probe. Lastly, the selection of internal spiked RNAs appropriate to our bacterial samples and useful for the data normalization step is presented.

Base Pair Mismatch↗
PubMed2006

Assessing incomplete deprotection of microarray oligonucleotides in situ.

En masse analysis of gene structure and function by array technologies will have a lasting and profound effect on biology and medicine. This impact can be compromised by low quality of probes within arrays, which we show can be caused by incomplete removal of chemical protecting groups. To solve this quality control problem, we present a sensitive, specific and facile method to detect these groups in situ on arrays using monoclonal antibodies and existing instrumentation. Screening of microarrays with these monoclonal antibodies should guide the consideration given to data derived from these and should enhance the accuracy of the results obtained.

Antibodies, Monoclonal↗
PubMed2006

Probe set algorithms: is there a rational best bet?

Affymetrix microarrays have become a standard experimental platform for studies of mRNA expression profiling. Their success is due, in part, to the multiple oligonucleotide features (probes) against each transcript (probe set). This multiple testing allows for more robust background assessments and gene expression measures, and has permitted the development of many computational methods to translate image data into a single normalized "signal" for mRNA transcript abundance. There are now many probe set algorithms that have been developed, with a gradual movement away from chip-by-chip methods (MAS5), to project-based model-fitting methods (dCHIP, RMA, others). Data interpretation is often profoundly changed by choice of algorithm, with disoriented biologists questioning what the "accurate" interpretation of their experiment is. Here, we summarize the debate concerning probe set algorithms. We provide examples of how changes in mismatch weight, normalizations, and construction of expression ratios each dramatically change data interpretation. All interpretations can be considered as computationally appropriate, but with varying biological credibility. We also illustrate the performance of two new hybrid algorithms (PLIER, GC-RMA) relative to more traditional algorithms (dCHIP, MAS5, Probe Profiler PCA, RMA) using an interactive power analysis tool. PLIER appears superior to other algorithms in avoiding false positives with poorly performing probe sets. Based on our interpretation of the literature, and examples presented here, we suggest that the variability in performance of probe set algorithms is more dependent upon assumptions regarding "background", than on calculations of "signal". We argue that "background" is an enormously complex variable that can only be vaguely quantified, and thus the "best" probe set algorithm will vary from project to project.

Algorithms↗
PubMed2004

Microarray-based AMASE as a novel approach for mutation detection.

Alterations in the p53 tumor suppressor gene are important events in many cases of human cancers. We have developed a novel microarray based approach for re-sequencing and mutation detection of the p53 gene. The method facilitates rapid and simple scanning of the target gene sequence and could be expanded to include other candidate cancer genes. The methodology employs the previously described apyrase-mediated allele-specific extension reaction (AMASE). In order to re-sequence the selected region, four extension oligonucleotides with different 3'-termini were used for each base position and they were covalently attached to the glass slide's surface. The amplified single-stranded DNA templates were then hybridized to the array followed by in situ extension with fluorescently labeled dNTPs in the presence of apyrase. The model system used was based on analysis of a 15 bp stretch in exon 5 of the p53 gene. Mutations were scored as allelic fractions calculated as (wt)/(wt + mut) signals. When apyrase was included in the extension reactions of wild type templates, the mean allelic fraction was 0.96. When apyrase was excluded with the same wild type templates, significantly lower allelic fractions were obtained. Two 60-mer synthetic oligonucleotides were used to establish the detectable amount of mutations with AMASE and a clear distinction between all the points could be made. Several samples from different stages of skin malignancies were also analyzed. The results from this study imply the possibility to efficiently and accurately re-sequence the entire p53 gene with AMASE technology.

Alleles↗