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Evaluation of suitable reference genes for gene expression studies in the developing mouse cortex using RT-qPCR
Ananya Uppalapati1, Timothy Wang1, Lena H Nguyen2
1Department of Neuroscience, School of Behavioral and Brain Sciences, The University of Texas at Dallas, Richardson, TX, 75080, USA.
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.
