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Background <p>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.</p> Results <p>Here, we evaluated the stability of five housekeeping genes, <i>Actb</i>, <i>Gapdh</i>, <i>B2m</i>, <i>Rpl13a</i>, and <i>Hprt</i>, 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 <i>B2m</i>,<i> Gapdh</i>, and <i>Hprt</i>, or a combination of <i>B2m/Gapdh</i> and <i>B2m/Hprt</i>, were the most stably expressed genes or gene pairs. In contrast, <i>Actb</i> and <i>Rpl13a</i> were the least stably expressed.</p> Conclusion <p>This study identifies <i>B2m</i>, <i>Gapdh</i>, and <i>Hprt</i> 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.</p>

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

  • 期刊论文

  • 出版模式：

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  • 发表日期：

    2025年2月18日

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  • Ananya Uppalapati,
  • Timothy Wang,
  • Lena H. Nguyen

摘要

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.