Reference-Gene Stability for RT-qPCR in the Medial Prefrontal Cortex and Hippocampal Subregions of the 5xFAD Mouse Brain

Abstract

Reverse transcription quantitative real-time PCR (RT-qPCR) requires normalization of the data to stably expressed reference genes, whose suitability must be validated for each experimental model and tissue. The present work aimed to assess the expression stability of seven candidate reference genes (Ppia, Ywhaz, Rpl27, Pgk1, Sdha, Gapdh, Actb) in three brain structures, the medial prefrontal cortex, the dorsal and ventral hippocampus, in 5xFAD mice, a model of Alzheimer's disease, and their wild-type controls. In addition to the selection of stable reference genes, we also propose combining reference genes into multiplex assays, which reduces the number of individual reactions while preserving normalization accuracy. mRNA levels were measured by RT-qPCR using hydrolysis probes, and expression stability was analyzed separately for each brain structure using the online tool RefFinder, which combines four algorithms (comparative Delta-Ct, BestKeeper, NormFinder and geNorm) to compute a comprehensive ranking. Reference gene stability proved to be region-specific. The most stable pairs were Ywhaz and Actb for the medial prefrontal cortex, Gapdh and Actb for the dorsal hippocampus, and Rpl27 and Pgk1 for the ventral hippocampus. Actb ranked among the most stable genes in the cortex and dorsal hippocampus but among the least stable in the ventral hippocampus, whereas Ppia was the least stable throughout. Expression of most candidate genes did not differ between wild-type and 5xFAD mice. The exception was Ywhaz, which was reduced in the ventral hippocampus of 5xFAD mice, consistent with its low stability ranking there. These results demonstrate the region-specific nature of reference gene selection in the 5xFAD mouse and allow us to recommend specific gene sets for RT-qPCR normalization in the medial prefrontal cortex and the dorsal and ventral hippocampus.