Background: Cross-species gene expression analyses using oligonucleotide microarrays designed to evaluate a single species can provide spurious results due to mismatches between the interrogated transcriptome and arrayed probes. Based on the most recent human and chimpanzee genome assemblies, we developed updated and accessible probe masking methods that allow human Affymetrix oligonucleotide microarrays to be used for robust genome-wide expression analyses in both species. In this process, only data from oligonucleotide probes predicted to have robust hybridization sensitivity and specificity for both transcriptomes are retained for analysis.

Results: To characterize the utility of this resource, we applied our mask protocols to existing expression data from brains, livers, hearts, testes, and kidneys derived from both species and determined the effects probe numbers have on expression scores of specific transcripts. In all five tissues, probe sets with decreasing numbers of probes showed non-linear trends towards increased variation in expression scores. The relationships between expression variation and probe number in brain data closely matched those observed in simulated expression data sets subjected to random probe masking. However, there is evidence that additional factors affect the observed relationships between gene expression scores and probe number in tissues such as liver and kidney. In parallel, we observed that decreasing the number of probes within probe sets lead to linear increases in both gained and lost inferences of differential cross-species expression in all five tissues, which will affect the interpretation of expression data subject to masking.

Conclusion: We introduce a readily implemented and updated resource for human and chimpanzee transcriptome analysis through a commonly used microarray platform. Based on empirical observations derived from the analysis of five distinct data sets, we provide novel guidelines for the interpretation of masked data that take the number of probes present in a given probe set into consideration. These guidelines are applicable to other customized applications that involve masking data from specific subsets of probes.