Unknown,Transcriptomics,Genomics,Proteomics

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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 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.

ORGANISM(S): Mus musculus

SUBMITTER: Luce Dauphinot

PROVIDER: E-GEOD-7574 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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On-chip hybridization kinetics for optimization of gene expression experiments.

Khomyakova Elena E Livshits Mikheil A MA Steinhauser Marie-Caroline MC Dauphinot Luce L Cohen-Kaminsky Sylvia S Rossier Jean J Soussaline Françoise F Potier Marie-Claude MC

BioTechniques 20080101 1

DNA microarray technology is a powerful tool for getting an overview of gene expression in biological samples. Although the successful use 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 ...[more]

PMID: 18254388

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Project description:Recombinant tissue-type plasminogen activator (tPA) is the fibrinolytic drug of choice to treat stroke patients. However, a growing body of evidence indicates that besides its beneficial thrombolytic role, tPA can also have a deleterious effect on the ischaemic brain. Although ageing influences stroke incidence, complications and outcome, age-dependent relationships between endogenous tPA and stroke injuries have not been investigated yet. Here, we report that ageing is associated with a selective lowering of brain tPA expression in the murine brain. Moreover, our results show that albumin D site-binding protein (DBP) as a key age-associated regulator of the neuronal transcription of tPA. Additionally, inhibition of DBP-mediated tPA expression confers in vitro neuroprotection. Accordingly, reduced levels of tPA in old mice are associated with smaller excitotoxic/ischaemic injuries and protection of the permeability of the neurovascular unit during cerebral ischaemia. Likewise, we provide neuroradiological evidence indicating the existence of an inverse relationship between age and the volume of the ischaemic lesion in patients with acute ischaemic stroke. Together, these results indicate that the relationship among DBP, tPA and ageing play an important role in the outcome of cerebral ischaemia. For each stage (4 months and 21 months), samples were prepared by pooling an equal amount of the 3 individual RNA. Five M-BM-5g of total RNA from each pool were reverse-transcribed in the presence of 7.5M-BM-5M random hexamers (GE Healthcare), 75M-BM-5M aminoallyl-dUTP (Sigma-Aldrich) and 100U Rtases Reverse-iT Blend (Thermo scientific) overnight at 37M-BM-0C. Aminoallyl cDNAs were then labelled with Cy5 or Cy3 mono-Reactive Dye (GE Healthcare) according to the manusfacturerM-bM-^@M-^Ys instructions. Labelled cDNAs were then purified on Qiaquick PCR prurification kit (Qiagen) and hybridized to the RNG-MRC_MM25k_EVRY microarrays (Le brigand et al., NAR, 2006) according to the RNG procedures (http://www.microarray.fr:8080/merge/index). Each pool was hybridized in duplicate dye-swap independent experiments. After hybridization, median signal and median background intensities were extracted using Genepix 4.1 software (Axon Instruments). We have first evaluated the level of expression of each gene by calculating the mean ratio M-bM-^@M-^\signal/backgroundM-bM-^@M-^] for each corresponding spot from the 2 dye-swap experiments. Genes were considered to be expressed if their mean ratio was higher or equal to 1.2. Genes with mean ratio < 1.2 in the 2 conditions were not used for further analysis. These filtered data were then submitted to VARAN (http//:www. bionet. espci.fr) for normalization by lowess fit and differential expression analysis (Golfier et al., 2004). Normalized log2ratio 21 months/4 months were then used for further statistical analysis by SAM software (Tusher et al., 2001) and t-test with a p-value equal to 5% using TIGR Multiexperiment Viewer (TM4:MeV, http://www.tm4.org/mev.html). The final list of differentially expressed genes was established by comparing the results obtained with the 3 methods.

Project description:The incidence of esophageal and junctional adenocarcinoma has increased 6-fold in the west in the last 30 years and 5 year survival remains <14%. We aimed to characterize genome-wide aberrations in esophageal adenocarcinoma to further understand disease pathogenesis and ultimately identify groups with differential survivals with implications for clinical management. Oligo-array-based high-resolution analysis of copy number changes in 89 fresh frozen esophageal adenocarcinoma resection sections with long-term clinical follow-up data was performed. Good quality array comparative genomic hybridization profiles were obtained from 56/89 of the samples (63%). Our analyses confirmed known genomic aberrations in esophageal adenocarcinoma and identified 3 novel genomic regions with copy number changes. A modified T test with adjusted Bonferroni correction identified 17 genes with significantly different log2 ratios (p<4x10-7), including 6 novel genes, that characterize a group with a poorer survival. Total RNA isolated from human tissue sections was used to make fluorescently labeled cRNA that was hybridized to DNA oligonucleotide. Briefly, 4 µg of total RNA was used to synthesize dsDNA through reverse transcription. cRNA was produced by in vitro transcription and labeled postsynthetically with Cy3 or Cy5. Two populations of labeled cRNA, a reference population and an experimental population, were compared with each other by competitive hybridization to microarrays. Two hybridizations were done with each cRNA sample pair using a fluorescent dye reversal strategy. Human microarrays contained oligonucleotide probes corresponding to approximately 21,000 genes. All oligonucleotide probes on the microarrays were synthesized in situ with inkjet technology (Agilent Technologies, Palo Alto, CA). After hybridization, arrays were scanned and fluorescence intensities for each probe were recorded. Ratios of transcript abundance (experimental to control) were obtained following normalization and correction of the array intensity data. Gene expression data analysis was done with the Rosetta Resolver gene expression analysis software (version 7.0, Rosetta Biosoftware, Seattle, WA).

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

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On-chip hybridization kinetics for optimization of gene expression experiments.

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