Evaluation of suitable reference genes for gene expression studies in the developing mouse cortex using RT-qPCR.

Background: Real-time quantitative PCR (RT-qPCR) is a widely used method to investigate gene expression in neuroscience studies. Accurate relative quantification of RT-qPCR requires the selection of reference genes that are stably expressed across the experimental conditions and tissues of interest. While RT-qPCR is often performed to investigate gene expression changes during neurodevelopment, few studies have examined the expression stability of commonly used reference genes in the developing mouse cortex.

Results: Here, we evaluated the stability of five housekeeping genes, Actb, Gapdh, B2m, Rpl13a, and Hprt, in cortical tissue from mice at embryonic day 15 to postnatal day 0 to identify optimal reference genes with stable expression during late corticogenesis. The expression stability was assessed using five computational algorithms: BestKeeper, geNorm, NormFinder, DeltaCt, and RefFinder. Our results showed that B2m, Gapdh, and Hprt, or a combination of B2m/Gapdh and B2m/Hprt, were the most stably expressed genes or gene pairs. In contrast, Actb and Rpl13a were the least stably expressed.

Conclusion: This study identifies B2m, Gapdh, and Hprt as suitable reference genes for relative quantification in RT-qPCR-based cortical development studies spanning the period of embryonic day 15 to postnatal day 0.