de Jong S et al. BMC Genomics 2010, 11:20 http://www.biomedcentral.com/1471-2164/11/20 We observed that brain region-specific effects can be detected independent of strain origin. Genetic variation is thought to be a major regulator of gene expression [16,18,39]; however, the differential expression profiles between the amygdala and hippocampus as observed in our study seem to represent natural variation driven by tissue-specific biological processes instead. The Red module especially differentiates between brain region samples, without any interference from genetic back- ground (Figure 2c-d). Previous studies investigating dif- ferences in brain regions also found that regional expression could be detected independent of strain. Whether genetic or tissue-specific effects have a stron- ger influence on gene expression is still under debate [40-44]. The results from our study suggest that the magnitude of the effect of strain and brain region is about the same, as module significance and eigengene values are comparable. However, since the module separating strains may not contain actual strain effects but be due to a hybridization artifact, it is likely that the effect of genetic strain differences is smaller in reality than the functional differences between tissues. Interac- tion effects between strain and brain region of all 13,627 probes were also assessed (see Additional File 7). This analysis revealed only 8 genes (Arsj, Baiap2l1, Fgf10, Myoc, Krt9, Lyd, 4930511J11Rik, Lypd1) of which differ- ential expression between amygdala and hippocampus was dependent on strain. Of these, Fgf10 and 4930511J11Rik belonged to the Red module. These small numbers suggests that interaction effects between strain and brain region are very limited. The current dataset provides a comparison between two brain region tissues only and is therefore somewhat limited in its perspective. While Red and Pink modules clearly separate amygdala and hippocampus in these two strains, the functions represented in these modules may extend to other (brain) regions as well. When we com- pared our findings with spinal cord data from the same individuals, only two of the ten modules were preserved in spinal cord (i.e. Magenta and Turquoise). As in the amygdala and hippocampus, the Magenta module again strongly differentiated between strains in spinal cord, suggesting that the SNP artifact in the probe sequences is driving the strain-specific differences throughout dif- ferent neuronal tissues. The Red and Pink module dif- ferentiating between amygdala and hippocampus, however, are not conserved in spinal cord and are there- fore more likely to be amygdala or hippocampus specific. The Magenta module is enriched for genes differen- tially expressed between strains independent of brain region. The module eigengene of this group of target genes was able to fully distinguish A/J and C57BL/6J samples (Figure 2a-b). Closer examination revealed that Page 9 of 14 a significant number of probe sequences of genes in the Magenta module contain nucleotide variants (SNPs) that differ between these two strains. The Illumina expres- sion arrays used in this study were developed using the C57BL/6J as the reference strain with probes optimally designed for the C57BL/6J genome. If there are SNP variants between strains within a probe sequence it is expected that C57BL/6J hybridization will be more effective. This prediction coincides with the systemati- cally lower expression levels of probes in the Magenta module in strain A/J (Figure 3). When the network is re-constructed after removing genes in the magenta module that were known to contain SNPs, the remain- ing magenta colored genes still fall into the same clus- ter. This module also still differentiates between strains. Therefore, it is possible that these probes represent real strain differences. However, resequencing of the probe sequences not containing a known SNP revealed that there are many unknown strain-specific polymorphisms in probe regions that may affect gene expression mea- surements between strains. The issue of SNP variants in probe regions causing hybridization artifacts has been described before when short (25-mer) cDNA probes were used [45,46], and more recently for long (60-mer) oligonucleotide probes [47]. In addition, it was found that mismatches do affect hybridization intensity, depending on the position of the SNP. By studying the enrichment of modules with regard to SNP-containing probes, our module-based analysis was able to detect this technical artifact. Further, we found that significantly increased numbers of SNP variants at the probe regions were also observed at loci with strong cis-effects in an eQTL analysis of the WTCCC heterogeneous stock database. A standard gene-based analysis could have easily missed this systematic bias. Co-expression modules may represent technical artifacts, tissue contaminations, or other biologically uninteresting perturbations. This is why functional enrichment analyses, module preserva- tion studies across array platforms, and other forms of validation are required to verify that co-expression mod- ules are indeed biologically meaningful. In our data, we found that a functional enrichment analysis for known gene ontologies did not find any significant findings for the Magenta module (which arose due to hybridization artifacts). We conclude that careful analysis of probe regions is warranted in an expression array study, espe- cially when only a limited number of (parental) inbred strains are involved. We expect that our findings also apply to transgenic mouse models in which part of the genomic sequence may still be from a different genetic background. The hybridization artifact is not present in the mod- ules found to be significantly enriched with genes