Quantitative PCR deconstruction of discrepancies between results reported by different hybridization platforms.

Differences in hybridization platforms used in gene array analysis experiments can lead to significant differences in hybridization results. In this study we used quantitative reverse transcription-polymerase chain reaction (qRT-PCR) to investigate discrepant results between the National Institute of Environmental Health Sciences cDNA and Affymetrix oligo platforms used to evaluate hepatic gene expression changes in rats exposed to methapyrilene. Caldesmon cDNA platform hybridization results showed decreases in gene expression levels for the high-dose methapyrilene 7-day pooled samples compared with their controls. By contrast, the Affymetrix oligonucleotide platform showed increases in expression levels for these samples. Quantitative gene expression measurements provide an explanation for the discrepancies observed for these samples. In the case of caldesmon, there is a 74-base sequence in the cDNA clone that is absent in the Affymetrix sequence. The amplicon based on the cDNA clone shows > 100-fold suppression relative to the day 7 high-dose methapyrilene-pooled control. These data demonstrate the importance of using a "gold standard," such as qRT-PCR to confirm key hybridization results as well as to understand the sources of discrepancies resulting from different hybridization platforms.