Customized PCR-array analysis informed by gene-chip microarray and biological hypothesis reveals pathways involved in...

Validation of gene-chip microarray results is one of the challenges in genomic studies. The successful use of a custom-designed 96-well polymerase chain reaction (PCR) array to study the unexpected inflammatory effect of environmental titanium dioxide (TiO2) particles on the lungs of pregnant mice, with similar results not seen in control mice, is reported. In our approach, selection of candidate genes for the custom PCR array was informed by prior gene-chip microarray profiling. Results demonstrated multiple upregulation of genes in the lungs of pregnant but not control mice produced by TiO2 exposure. Customized PCR array is a flexible tool that offers the ability to combine the "blind" genome-wide scan with a hypothesis-driven approach, by including both the "candidate" genes for validation positively identified by the microarray and biologically relevant "suspects" that failed to be found in the genomic data. Compared to conventional gene-by-gene qPCR or manufacturer-preset pathway kits, this technique provides a cost-effective and time-saving method of analysis and allows for a strong, easily detectable signal. Genes with confirmed differential expression were further used for pathway analysis and indicated involvement in several biologically relevant pathways including allergy mediator signaling in dendritic cells. Finally, an analytical network was created that will inform further mechanistic studies. The dual purpose of the work was to demonstrate that the novel custom PCR array is a convenient approach to validate the microarray results, and to obtain biologically significant data on TiO2-induced inflammation by following the PCR array with pathway analysis, which provided feasible hypotheses to support future experimental studies.