Effect of RNA concentration in cross-species hybridization
Ontology highlight
ABSTRACT:
ORGANISM(S): Marmota monax Homo sapiens
PROVIDER: GSE437 | GEO | 2003/05/27
SECONDARY ACCESSION(S): PRJNA85015
REPOSITORIES: GEO
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Project description:The objective of this work was to determine the effectiveness of cross-hybridization of gDNA from five native soil nematodes to an Affymetrix Caenorhabditis elegans tiling array. Cross-hybridization experiments using C. briggsae, for which genome information is available, allowed hybridisation intensities to be correlated with known sequence differences. Initial analysis of data by conventional array-based Comparative Genomic Hybridization (aCGH) techniques at the chip level lead to misleading results due to an artefact from the combination of scaling, bandwidth smoothing, and differential GC content in exon and intron regions. To circumvent this artefact, individual probes were instead normalized and centered by adjusting for probe-specific thermodynamic binding affinity. However, cross-hybridization of C. briggsae DNA revealed that the resultant probe intensities alone were still uncorrelated to sequence similarity below 90% identity. Below 90% similarity, all probes hybridize uniformly poorly, and above 90% similarity the hybridization differences are not large enough to detect over background, therefore, no 'threshold' ratio of hybridization intensity was successful at identifying probes with similarity to the heterologous genome. In light of the observations described here, we suggest that the criteria for replication and verification of gene expression profiles generated from cross-species microarray hybridizations be more stringent than typically adopted for con-specific hybridizations. Genomic DNA from Caenorhabditis elegans N2 (Bristol), C. elegans CB4856 (Hawaiian), C. briggsae AF16, Oscheius tipulae KS585, Oscheius FVV-2 KS555, Mesorhabditis sp. KS587, Acrobeloides sp. KS586, and Chiloplacus sp. KS584 were hybridized onto C. elegans Affymetrix tiling array (two replicate chips were performed for each species).
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