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Paralog Specificity in cDNA Microarray Experiments

ABSTRACT: cDNA microarrays have become the technology of choice for profiling the expression of thousands of genes simultaneously in a single sample. However, cross-hybridization between targets and probes that do not correspond must be considered carefully during the design, analysis, and interpretation of these experiments. Here, we examined this potentially confounding issue by competitively hybridizing fragments with similar sequence obtained from two paralogous genes to a custom cDNA microarray that contains probes for only one of these two genes. We found that although a large contiguous portion of these two fragments was 84% identical and only one target was represented on the array, there was very little cross-hybridization of the non-represented target to either the paralogous probes or other (non-similar) features. We conclude that cDNA microarrays are indeed very specific and that promiscuous paralog hybridization adds little noise to the data. More generally, the absence of specific probes for any given gene does not increase the chance for cross-hybridization between non-specific probes and targets, even if sequence similarity is relatively high.

ORGANISM(S): Haplochromis burtoni

PROVIDER: GSE16939 | GEO | 2009/07/03

SECONDARY ACCESSION(S): PRJNA117655

REPOSITORIES: GEO

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Project description:The purpose of this microarray experiment was to validate the Del-Mar 14K Chicken Integrated Systems Microarray for different chicken tissues and to determine the utility of this chicken cDNA microarray for other closely related and more distant avian species. The Del-Mar 14 K array was constructed from cDNAs amplified from EST clones sequenced from five normalized chicken cDNA libraries derived from neuroendocrine (5,929), abdominal fat (4,800), liver (2,635), skeletal muscle (2,398), reproductive tract (2,008), 387 long (70mer) oligonucleotides and 72 quality control spots. The array contains 17,770 cDNA clones, where protein matches were found by BlastX analysis for 68% of chicken contigs and 46% of singleton sequences represented on the array. A reference RNA design was used for the hybridization of total RNA from four chicken tissues (liver, abdominal fat, breast muscle and hypothalamus) and the cross-species hybridization (CSH) of total RNA from tissue from birds representing four orders of the Class Aves [Galliformes (chicken, Coturnix quail and domestic turkey), Anseriformes (Peking duck), Falconiformes (American kestrel) and Passeriformes (American tree sparrow)]. A reference RNA pool was made from an equal amount of high-quality total RNA extracted from chicken liver, abdominal fat, breast muscle and hypothalamus. Each of the 43 microarrays was co-hybridized with Cy3-labeled cDNA targets from one of the avian tissue samples and Cy5-labled cDNA targets from the reference chicken RNA pool. Loess-normalized data were used to determine the number of cDNAs expressed in chicken tissues and the number of genes (cDNAs) detectable by cross-hybridization with various avian tissue samples. The Cy5-labeled reference samples were used to determine the coefficient of variation across the 43 microarrays. This study shows a remarkably high level of cross hybridization of Cy3-labeled cDNA targets from a wide range of avian species to the Del-Mar 14K microarray, where 38 to 62% of the cDNA probes on the chicken array (genes) were detectable. Keywords: Transcriptional profiling, Del-Mar 14K Chicken Integrated Systems Microarray validation, multi-tissues, cross-species hybridization, class Aves

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Paralog Specificity in cDNA Microarray Experiments

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