Selection of Appropriate Reference Genes in Apoe-/- Mouse Brain

Date

2026-04-01

Author

Mengi, Naz
Yanık, Tülin
Adams, Michelle

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BackgroundReverse 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 MethodTo address this problem, expression stability of six frequently used reference genes:Actb,Gapdh,Rpl13a,Rplp0,Hprt1, and Ywhazin 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.ResultsResults 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,Hprt1in the cerebral cortex andActbin the hippocampus showed significant changes by diet and genotype.Comparison with existing methodsReference 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.ConclusionThese results underline the importance of validating gene stability under specific experimental conditions.

URI

https://hdl.handle.net/11511/119365

Collections

Department of Biology, Article