Selection of appropriate reference genes in Apoe-/- mouse brain

Background: Reverse transcription quantitative real-time polymerase chain reaction is the gold standard for gene expression quantification. Yet, this method's accuracy heavily depends on choosing appropriate reference genes for data normalization. Reference genes must display stable expression levels across biological and experimental conditions to ensure accurate and meaningful results. New method: mice given a chow or Western diet was evaluated using RefFinder, which utilizes four commonly used algorithms: the comparative ∆Ct method, BestKeeper, NormFinder, and geNorm. Additionally, the geometric mean of the two most stable genes was used to normalize the expression of the others to test the variability of less stable genes across brain regions, genotypes, and dietary conditions. Results: in the hypothalamus showed significant changes by diet and genotype. Comparison with existing methods: The combined RefFinder ranking provided a more robust evaluation, emphasizing subtle differences in gene stability across experimental conditions for more accurate reference gene selection Conclusion: These results underline the importance of validating gene stability under specific experimental conditions.