Reference Gene Validation in the Brain Regions of Young Rats after Pentylenetetrazole-Induced Seizures

Reverse transcription followed by quantitative polymerase chain reaction (qRT-PCR) is a powerful and commonly used tool for gene expression analysis. It requires the right choice of stably expressed reference genes for accurate normalization. In this work, we aimed to select the optimal reference genes for qRT-PCR normalization within different brain areas during the first week following pentylenetetrazole-induced seizures in immature (P20-22) Wistar rats. We have tested the expression stability of a panel of nine housekeeping genes:Actb, Gapdh, B2m,Rpl13a, Sdha, Ppia,Hprt1, Pgk1,andYwhaz.Based on geometric averaging of ranks obtained by four common algorithms (geNorm, NormFinder, BestKeeper, Comparative Delta-Ct), we found that the stability of tested reference genes varied significantly between different brain regions. The expression of the tested panel of genes was very stable within the medial prefrontal and temporal cortex, and the dorsal hippocampus. However, within the ventral hippocampus, the entorhinal cortex and amygdala expression levels of most of the tested genes were not steady. The data revealed that in the pentylenetetrazole-induced seizure model in juvenile rats,Pgk1,Ppia, andB2mexpression are the most stable within the medial prefrontal cortex;Ppia,Rpl13a, andSdhawithin the temporal cortex;Pgk1,Ppia, andRpl13awithin the entorhinal cortex;Gapdh,Ppia, andPgk1within the dorsal hippocampus;Rpl13a,Sdha,andPpiawithin the ventral hippocampus; andSdha,Pgk1, andPpiawithin the amygdala. Our data indicate the need for a differential selection of reference genes across brain regions, including the dorsal and ventral hippocampus.