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A systematic spatial bias in microarray hybridizations caused by probe spot position-dependent variability in lateral diffusion

ABSTRACT: The hybridization of nucleic acid targets with surface-immobilized probes is a widely used assay format for high-throughput detection of many parallel targetsin medical and biological research. Though commonly applied, microarray technology still suffers limitations arising from problems of data robustness and reproducibility across platforms, stemming in part from an incomplete understanding of the complex processes governing surface hybridization behavior. It has been observed that there are non-random spatial variations within individual microarray hybridizations, but the causative mechanisms of positional bias remain largely unexplained. This study identifies a symptomatic spatial bias in surface hybridization signal intensitywith systematically increased signal intensities of spots located at the boundaries of the spotted areas of the microarray slide and characterizes the underlying mechanistic principle of this bias using a simplified block array format. Experimentally-derived hybridization dynamics are compared with a mathematical modeling analysis, which together showthat the driver of the spatial bias is a position-dependent variation in lateral diffusion. Numerical simulations employing a diffusion-based model are used to demonstrate the strong influence of microarray well geometry on this spatial bias and to determine optimal conditions for which this bias can be minimized or eliminated, resulting in increased uniformity of microarray hybridization.

ORGANISM(S): Candidatus Protochlamydia amoebophila UWE25 Pseudodesulfovibrio halophilus

PROVIDER: GSE26275 | GEO | 2010/12/22

SECONDARY ACCESSION(S): PRJNA135141

REPOSITORIES: GEO

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Project description:Background Coral reefs belong to the most ecologically and economically important ecosystems on our planet. Yet, they are under steady decline worldwide due to rising sea surface temperatures, disease, and pollution. Understanding the molecular impact of these stressors on different coral species is imperative in order to predict how coral populations will respond to this continued disturbance. The use of molecular tools such as microarrays has provided deep insight into the molecular stress response of corals. Here, we have performed comparative genomic hybridizations (CGH) with different coral species to an Acropora palmata microarray platform containing 13,546 cDNA clones in order to identify potentially rapidly evolving genes and to determine the suitability of existing microarray platforms for use in gene expression studies (via heterologous hybridization). Results Our results showed that the current microarray platform for A. palmata is able to provide biological relevant information for a wide variety of coral species covering both the complex clade as well the robust clade. Analysis of the fraction of highly diverged genes showed a significantly higher amount of genes without annotation corroborating previous findings that point towards a higher rate of divergence for taxonomically restricted genes. Among the genes with annotation, we found many mitochondrial genes to be highly diverged in M. faveolata when compared to A. palmata, while the majority of nuclear encoded genes maintained an average divergence rate. Conclusions The use of present microarray platforms for transcriptional analyses in different coral species will greatly enhance the understanding of the molecular basis of stress and health and highlight evolutionary differences between scleractinian coral species. On a genomic basis, we show that cDNA arrays can be used to identify patterns of divergence. Mitochondrion-encoded genes seem to have diverged faster than nuclear encoded genes in robust corals. Accordingly, this needs to be taken into account when using mitochondrial markers for scleractinian phylogenies. Microarray experiments were performed as follows. Appropriate Cy3 and Cy5 labeled DNAs were mixed together in a hybridization buffer containing 0.25% SDS, 25 mM HEPES and 3 × SSC, resulting in a final volume of 70?l. The hybridization mixtures were boiled for 2 min at 99°C and allowed to cool at room temperature for 5 min. The cooled hybridization mixtures were pipetted under an mSeries Lifterslip (Erie Scientific), and hybridization took place in Corning hybridization chambers overnight at 55°C. Microarrays were washed once in 2 × SSC, 0.03% SDS heated to 55 °C for 5 min. followed by one wash in 1 x SSC and another wash in 0.2 x SSC for 5 min each. The slides were kept in 0.2 × SSC until analysis. Slides were dried via centrifugation and scanned using an Axon 4000B scanner. The experimental setup followed a reference design, i.e., all samples were hybridized against the same pool of labeled A. palmata DNA. For each species, a total of four hybridizations were performed, including two dye swap hybridizations in order to account for potential dye bias i.e. two hybridizations with Cy3 labeled M. faveolata DNA against a Cy5 labeled A. palmata reference and two hybridizations with Cy5 labeled M. faveolata DNA against a Cy3 labeled Cy3 A. palmata reference were performed. The same hybridization scheme was used for A. cervicornis and S. radians.

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A systematic spatial bias in microarray hybridizations caused by probe spot position-dependent variability in lateral diffusion

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