State Key Laboratory of Cardiovascular Disease, Fuwai Hospital, National Center for Cardiovascular Diseases, Chinese Academy of Medical Sciences and Peking Union Medical College, 167A Beilishi Road, Xi Cheng District, Beijing, 100037, People's Republic of China.
BMC Dev Biol. 2021 Sep 8;21(1):13. doi: 10.1186/s12861-021-00244-6.
Real-time quantitative polymerase chain reaction (RT-qPCR) is a widely-used standard assay for assessing gene expression. RT-qPCR data requires reference genes for normalization to make the results comparable. Therefore, the selected reference gene should be highly stable in its expression throughout the experimental datasets. So far, reports about the optimal set of reference genes in murine left ventricle (LV) across embryonic and postnatal stages are few. The objective of our research was to identify the appropriate reference genes in murine LV among different developmental stages.
We investigated the gene expression profiles of 21 widely used housekeeping genes in murine LV from 7 different developmental stages (almost throughout the whole period of the mouse lifespan). The stabilities of the potential reference genes were evaluated by five methods: GeNorm, NormFinder, BestKeeper, Delta-Ct and RefFinder.
We proposed a set of reliable reference genes for normalization of RT-qPCR experimental data in different conditions. Furthermore, our results showed that 6 genes (18S, Hmbs, Ubc, Psmb4, Tfrc and Actb) are not recommended to be used as reference genes in murine LV development studies. The data also suggested that the Rplp0 gene might serve as an optimal reference gene in gene expression analysis.
Our study investigated the expression stability of the commonly used reference genes in process of LV development and maturation. We proposed a set of optimal reference genes that are suitable for accurate normalization of RT-qPCR data in specific conditions. Our findings may be helpful in future studies for investigating the gene expression patterns and mechanism of mammalian heart development.
实时定量聚合酶链反应（RT-qPCR）是一种广泛用于评估基因表达的标准检测方法。RT-qPCR 数据需要参考基因进行归一化处理，以使结果具有可比性。因此，所选的参考基因在整个实验数据集的表达中应该高度稳定。迄今为止，关于胚胎期和出生后阶段小鼠左心室（LV）中最佳参考基因集的报道很少。我们研究的目的是在不同发育阶段鉴定小鼠 LV 中合适的参考基因。
我们研究了 21 种广泛使用的管家基因在来自 7 个不同发育阶段的小鼠 LV 中的基因表达谱（几乎涵盖了小鼠整个生命周期）。通过 GeNorm、NormFinder、BestKeeper、Delta-Ct 和 RefFinder 五种方法评估潜在参考基因的稳定性。
我们提出了一套可靠的参考基因，用于在不同条件下归一化 RT-qPCR 实验数据。此外，我们的结果表明，在小鼠 LV 发育研究中，6 个基因（18S、Hmbs、Ubc、Psmb4、Tfrc 和 Actb）不建议作为参考基因使用。数据还表明，Rplp0 基因可能是基因表达分析的最佳参考基因。
本研究调查了 LV 发育和成熟过程中常用参考基因的表达稳定性。我们提出了一组最佳参考基因，适用于特定条件下 RT-qPCR 数据的准确归一化。我们的发现可能有助于未来研究哺乳动物心脏发育的基因表达模式和机制。
