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

Area of Science:

• Neuroscience
• Molecular Biology
• Genetics

Background:

• Quantitative real-time polymerase chain reaction (RT-qPCR) is a standard for gene expression analysis.
• Accurate gene expression quantification relies on selecting stable reference genes for normalization.
• Reference genes must exhibit consistent expression across diverse biological and experimental conditions.

Purpose of the Study:

• To evaluate the expression stability of six common reference genes.
• To identify the most stable reference genes for normalization in specific mouse brain regions, genotypes, and diets.
• To compare the performance of different algorithms for reference gene stability assessment.

Main Methods:

• Expression stability of *Actb*, *Gapdh*, *Rpl13a*, *Rplp0*, *Hprt1*, and *Ywhaz* was assessed in mouse cerebral cortex, hippocampus, and hypothalamus.
• Mice included C57BL/6 and Apoe⁻/⁻ genotypes fed either a chow or Western diet.
• RefFinder, integrating ∆Ct, BestKeeper, NormFinder, and geNorm algorithms, was used for comprehensive stability analysis.

Main Results:

• Reference gene stability varied significantly across brain regions, genotypes, and diets.
• The hypothalamus exhibited the least stable reference gene expression.
• Specific genes like *Hprt1* (cerebral cortex) and *Actb* (hypothalamus) showed significant diet and genotype-dependent expression changes.

Conclusions:

• Reference gene stability is condition-dependent and requires validation for specific experimental settings.
• A comprehensive approach using RefFinder provides a more robust evaluation of reference gene stability.
• Accurate gene expression normalization necessitates careful selection of validated reference genes.