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

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.

To address this problem, expression stability of six frequently used reference genes: Actb, Gapdh, Rpl13a, Rplp0, Hprt1, and Ywhaz in the cerebral cortex, hippocampus, and hypothalamus of C57BL/6 and Apoe^⁻/⁻ 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 demonstrated that reference genes were the least stable in the hypothalamus, and the comprehensive ranking of the reference genes differed between the cerebral cortex and the hypothalamus. Notably, Hprt1 in the cerebral cortex and Actb in the hypothalamus showed significant changes by diet and genotype.

Reference gene stability is often assessed using individual algorithms like ∆Ct, BestKeeper, NormFinder, or geNorm. These algorithms utilize mathematical models and assumptions . The combined RefFinder ranking provided a more robust evaluation, emphasizing subtle differences in gene stability across experimental conditions for more accurate reference gene selection .

These results underline the importance of validating gene stability under specific experimental conditions.