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Analysis of on-chip hybridization kinetics for optimization of gene expression experiments.

ABSTRACT: DNA microarray technology is a powerful tool for getting the overview of gene expression in biological samples. Although the successful application of microarray-based expression analysis was demonstrated in a number of applications, the main problem with this approach is the fact that expression levels deduced from hybridization experiments do not necessarily correlate with RNA concentrations. Moreover, oligonucleotide probes corresponding to the same gene can give different hybridization signals. Apart from cross-hybridizations and differential splicing, this could be due to secondary structures of probes or targets. In addition, for low copy genes, hybridization equilibrium may be reached after hybridization times much longer than the one commonly used (overnight, 15 hours). Thus, hybridization signals could depend on kinetic properties of the probe, which may vary between different oligonucleotide probes immobilized on the same microarray. To validate these hypothesis, on-chip hybridization kinetics and duplexes thermostability analysis were performed using oligonucleotide microarrays containing 50-mer probes corresponding to mouse genes. We demonstrate that differences in hybridization kinetics between the probes can influence the interpretation of expression data. Ways to improve the reliability of microarray-based expression analysis are discussed. Keywords: kinetics, gene expression, microarrays

ORGANISM(S): Mus musculus

PROVIDER: GSE7574 | GEO | 2007/09/30

SECONDARY ACCESSION(S): PRJNA100349

REPOSITORIES: GEO

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Analysis of on-chip hybridization kinetics for optimization of gene expression experiments.

Project description:DNA microarray technology is a powerful tool for getting the overview of gene expression in biological samples. Although the successful application of microarray-based expression analysis was demonstrated in a number of applications, the main problem with this approach is the fact that expression levels deduced from hybridization experiments do not necessarily correlate with RNA concentrations. Moreover, oligonucleotide probes corresponding to the same gene can give different hybridization signals. Apart from cross-hybridizations and differential splicing, this could be due to secondary structures of probes or targets. In addition, for low copy genes, hybridization equilibrium may be reached after hybridization times much longer than the one commonly used (overnight, 15 hours). Thus, hybridization signals could depend on kinetic properties of the probe, which may vary between different oligonucleotide probes immobilized on the same microarray. To validate these hypothesis, on-chip hybridization kinetics and duplexes thermostability analysis were performed using oligonucleotide microarrays containing 50-mer probes corresponding to mouse genes. We demonstrate that differences in hybridization kinetics between the probes can influence the interpretation of expression data. Ways to improve the reliability of microarray-based expression analysis are discussed. Keywords: kinetics, gene expression, microarrays Total RNA were extracted from 5 thymus and 6 cortex of B10D2 mice. For each tissue, equal amounts of each RNA were pooled and 15M-BM-5g were labeled by reverse-transcription and hybridized on neurotransmission microarrays. Three replicates of each experiment have been performed.

2007-09-29 | E-GEOD-7574 | biostudies-arrayexpress

Zoarces viviparus evaluation data

Project description:A 50-mer oligonucleotide microarray was designed for large-scale gene expression analysis in Z. viviparus. To measure the expression of the probes and the corresponding assembled transcripts, the pool of mRNA used for the sequencing was hybridized.

2009-07-24 | GSE14957 | GEO

Evidence of transcription in intergenic sequences in Rickettsia conorii

Project description:We used a microarray covering the whole genome of R. conorii to check if intergenic sequences were found transcribed. We checked the expression signals for probes corresponding to spacers as compared to probes corresponding to Open Reading Frames (ORFs). We got total RNA from R. conorii XTC cultures; we performed cDNA synthesis and then hybridizations. The hybridizations were repeated four times, and data were compared to check the reproducibility.

2011-09-27 | GSE24157 | GEO

Design and validation of microarray for chickpea (Cicer arietinum) based on the analysis of transcriptome data [gene expression]

Project description:Microarrays have increasingly become a powerful tool for high throughput gene-expression studies and discovery of novel biomarker genes. Developed for a large number of organisms, including plants, microarrays are commonly performed for species that have sequenced data, for performing gene expression analysis, miRNA profiling, comparative genomic hybridization (CGH), ChIP-on-chip and SNP analysis. Genomic resources are still very limited for chickpea, a very important food legume crop. Here, we report the design and comprehensive validation of Next Generation Sequencing transcriptome data for chickpea through microarray technology to develop a high-throughput resource for studying the expression of all the transcripts in different biological samples to help functional genomics and breeding programs. This microarray design was developed and validated jointly by Genotypic Technology Private Limited and National Institute of Plant Genome Research. First, we designed 400k probes using reads covering 35k assembled contigs and 100k singletons chickpea transcripts. The 400k chip was hybridized with DNA and RNA samples of chickpea and microarray analysis was carried out. A total of 73,922 probes were found to be specific to chickpea transcripts. Best probes were filtered from the analyzed data and a total of 61,659 probes were selected to develop the final microarray design in 60k gene-expression microarray format. The probes represented 51,444 unique transcripts. The probes were annotated based on their corresponding chickpea transcript and similarity with other plants species. Microarray results were concordant with previous results from the NGS studies. The design of custom oligonucleotide probes for microarrays have varied functional genomic applications and this approach represents a valuable resource for chickpea.

2017-01-07 | GSE78986 | GEO

Design and validation of microarray for chickpea (Cicer arietinum) based on the analysis of transcriptome data [CGH]

2017-01-07 | GSE78985 | GEO

Design and validation of 8X60k gene-expression microarray for chickpea (Cicer arietinum) based on the transcriptome data

2017-01-05 | GSE76972 | GEO

Oligonucleotide microarrays for the detection and identification of viable beer spoilage bacteria

Project description:A prototype oligonucleotide microarray was designed to detect and identify viable bacterial species with the potential to grow of common beer spoilage microorganisms from the genera Lactobacillus, Megasphaera, Pediococcus and Pectinatus. Probes targeted the intergenic spacer regions (ISR) between 16S and 23S rRNA, which were amplified in a combination of reverse transcriptase (RT) and polymerase chain reaction (PCR) prior to hybridization. This method allows the detection and discrimination of single bacterial species in a complex sample. Furthermore, microarrays using oligonucleotide probes targeting the ISR allow the distinction between viable bacteria with the potential to grow and non-growing bacteria. The results demonstrate the feasibility of oligonucleotide microarrays as a contamination control in food industry for the detection and identification of spoilage microorganisms within mixed population. Keywords: microarray, oligonucleotide, species-specific, detection, beer spoilage bacteria

2007-05-23 | GSE7840 | GEO

Determine the extent of inter-species hybridization in adult parasites.

Project description:We report the design, fabrication, and validation of a microarray covering the majority of the Schistosoma japonicum and Schistosoma mansoni transcriptomes. The oligonucleotide microarray contains 12,166 S. mansoni and 7055 S. japonicum target sequences. A confidence threshold of 0.001 (P value) was used in all analyses. The cross hybridization potential of the microarray was representative of 10,362 probes detected in both channels, while 12,052 probes hybridised to S. japonicum and 11,254 probes hybridised to S. mansoni. Differential hybridisation consisted of 3422 probes to S. mansoni mRNA and 3103 probes to S. japonicum mRNA. Important biological functions, such as transport, metabolism, immune evasion and host?parasite immunological interplay, cell communication, and sexual maturation are represented on this microarray. This is the first microarray commercially manufactured for studying schistosomes, and the large size and verified quality of the resource demonstrate its power for characterising the schistosome transcriptome. Keywords: Interspecies Comparison

2006-07-07 | GSE5191 | GEO

HyPro-MS analysis of proteins proximal to nuclear noncoding RNAs in HeLa cells

Project description:The ability of RNAs to form specific contacts with other macromolecules provides an important mechanism for subcellular compartmentalization. We developed a suite of hybridization-proximity (HyPro) labeling technologies for unbiased discovery of proteins (HyPro-MS) and transcripts (HyPro-seq) associated with RNAs of interest in genetically unperturbed cells. To generate the HyPro-MS dataset reported here, fixed and permeabilized HeLa cells were hybridized with digoxigenin-labeled oligonucleotide probes against noncoding RNAs 45S, NEAT1 or PNCTR and proteins co-localizing with these RNAs were biotinylated in situ using a custom-engineered HyPro enzyme containing a digoxigenin-binding domain. Biotinylated proteins were then captured on streptavidin beads and analyzed by LC-MS/MS.

2021-11-08 | PXD025264 | Pride

A custom genome-wide Plasmodium vivax 8x15K microarray for expression profiling of Indian clinical isolates showing complicated malaria

Project description:High density oligonucleotide microarrays have been used on Plasmodium vivax field isolates to estimate whole genome expression. However, no microarray platform has been experimentally optimized for studying the transcriptome of field isolates. In the present study, we adopted both bioinformatics and experimental testing approaches to select best optimized probes suitable for detecting parasite transcripts from field samples and included them in designing a custom 15K P. vivax microarray. This microarray has long oligonucleotide probes (60mer) that were in-situ synthesized onto glass slides using Agilent SurePrint technology and has been developed into an 8X15K format (8 identical arrays on a single slide). Probes in this array were experimentally validated and represents 4180 P. vivax genes in sense orientation, of which 1219 genes have also probes in antisense orientation. Validation of the 15K array by using field samples (n=14) has shown 99% of parasite transcript detection from any of the samples. Correlation analysis between duplicate probes (n=85) present in the arrays showed perfect correlation (r2=0.98) indicating the reproducibility. Multiple probes representing the same gene exhibited similar kind of expression pattern across the samples (positive correlation, r≥0.6). Comparison of hybridization data with the previous studies and quantitative real-time PCR experiments were performed to highlight the microarray validation procedure. This array is unique in its design, and results indicate that the array is sensitive and reproducible. Hence, this microarray could be a valuable functional genomics tool to generate reliable expression data from P. vivax field isolates.

2016-10-18 | GSE55644 | GEO

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