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Lacking Parakeet and Opossum Microarrays, Researchers Try Cross-Species Hybridization

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When University of Idaho biologist Kristen Carlberg wanted to study the effect of oxidative stress on budgerigar, or parakeet, genes in comparison to Japanese quail genes, she couldn’t call up Affymetrix and order some prefabricated GeneChips for overnight delivery. “Nobody has microarrays made with budgerigar genes,” Carlberg said in a presentation to the second annual Northwest Microarray Conference in Seattle.

So Carlberg tried what a small but growing number of microarray researchers are doing: She performed cross-species hybridization on microarrays spotted down with gene probes from a different organism.

In Carlberg’s case, the arrays were 3,000-spot chicken gene chips that Paul Neiman of the Fred Hutchison Cancer Research Center fabricated using chicken B and T cells. Chicken and budgerigar are as little as 83 percent homologous, and Carlberg was using fibroblast tissue from the budgie, not brain tissue. “It was the wrong species, and the wrong tissue,” she said. But the experiment worked.

The experiment involved finding changes in gene expression between budgerigars exposed to oxidative stress and those that were not, then changes in gene expression between quails exposed to oxidative stress and unexposed ones, and comparing the changes between the two organisms.

In this experiment, the cross-species effect would operate as a constant because each species would have a baseline hybridization rate, and the experiment would just measure change from that hybridization rate.

Carlberg further found that when she hybridized her budgie genes to the chicken array, the signal was not only greater than background, it almost matched in intensity the signal produced by chicken brain tissue mRNA. She was able to subsequently locate a number of genes that were over- or under-expressed in budgerigar cells but not quail cells, indicating a number of gene targets for the response of the budgerigar to oxidative stress.

Not only did this experiment yield valuable data about the genes implicated in oxidative stress response. It also showed Carlberg that “in some cases where you don’t have the right kind of array, you can use the cross-species array,” she said.

Carlberg plans to redo the experiment by hybridizing the mRNA from the exposed and unexposed birds to an 18,000-probe human cDNA array. The experiment is part of a larger study on budgerigar oxidative stress response, which is thought to contribute to the species’ longevity in comparison to other birds.

 

Mysterious Opossum Genome

Zhiqiang Wang of the Southwest Foundation for Biomedical research in San Antonio, Texas, has also tried the cross-species hybridization approach, to study melanoma-related gene expression in the opossum, Monodelphis domestica. This species is the only experimental model in which melanoma can be induced by expression to UV radiation alone, but its genome remains a black box.

Still, Wang thought it would be useful to hybridize mRNA from melanoma tumor tissues in the opossum to the DermArray, a 4,400-gene cDNA human array specifically created for the study of melanoma and other skin conditions. Wang purified RNA from a highly metastatic cell line from an opossum tumor, as well as a cell line from a benign tumor, and compared gene expression patterns in these two. Like Carlberg’s experiment, this design made the cross-species element a constant, since both the metastatic and non-metastatic lines would conceivably have the same baseline rate of hybridization to the human array.

Curiously, Wang found that his hybridizations actually produced a stronger signal than those in side-by-side human array experiments.

Wang’s advice to other scientists who would like to try cross-species hybridization is “first try the hybridizations using the same stringency” as recommended for the species spotted on the array. “ f it does not work as well as the same species, do not lower the hybridization temperature. Just lower the washing conditions.”

These opossum microarray experiments produced 109 differentially expressed genes, 25 of which Wang isolated as “candidate genes for melanoma growth and metastasis, based on comparisons to previous research involving their human homologs and melanoma. He is planning to sequence these genes.

— MMJ

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