Genetics Division, Department of Morphology and Genetics, Universidade Federal de São Paulo, São Paulo, Brazil.
Laboratory of Developmental Biology, Department of Morphology and Genetics, Universidade Federal de São Paulo, São Paulo, Brazil.
Gene. 2019 Aug 20;710:148-155. doi: 10.1016/j.gene.2019.05.042. Epub 2019 Jun 2.
qRT-PCR requires reliable internal control genes stably expressed in different samples and experimental conditions. The stability of reference genes is rarely tested experimentally, especially in developing tissues given the singularity of these samples. Here we evaluated the suitability of a set of reference genes (Actb, Gapdh, Tbp, Pgk1 and Sdha) using samples from early mouse embryo tissues that are widely used in research (somites, prosencephalon and heart) at different developmental stages. The comparative ΔCq method and five software packages (NormFinder, geNorm, BestKeeper, DataAssist and RefFinder) were used to rank the most stable genes while GenEx and GeNorm programs determined the optimal total number of reference genes for a reliable normalization. The ranking of most reliable reference genes was different for each tissue evaluated: (1) in somite from embryos with 16-18 somite pairs stage, the combination of Pgk1 and Actb provided the best normalization and Actb also presented high stability levels at an earlier developmental stage; (2) Gapdh is the most stable gene in prosencephalon in the two developmental stages tested; and (3) in heart samples, Sdha, Gapdh and Actb were the best combination for qPCR normalization. The analysis of these three tissues simultaneously indicated the combination of Gapdh, Actb and Tbp as the most reliable internal control. This study highlights the importance of appropriate reference genes according to the cell type and/or tissue of interest. The data here described can be applied in future research using mouse embryos as a model for mammalian development.
qRT-PCR 需要在不同的样本和实验条件下稳定表达的可靠内参基因。参考基因的稳定性很少在实验中进行测试，尤其是在发育组织中，因为这些样本具有特殊性。在这里，我们评估了一组参考基因（Actb、Gapdh、Tbp、Pgk1 和 Sdha）在不同发育阶段的早期小鼠胚胎组织样本中的适用性（体节、前脑和心脏），这些样本在研究中被广泛使用。我们使用比较 ΔCq 方法和五个软件包（NormFinder、geNorm、BestKeeper、DataAssist 和 RefFinder）对最稳定的基因进行排名，同时使用 GenEx 和 GeNorm 程序确定可靠归一化的最佳参考基因总数。最可靠的参考基因的排名因评估的组织而异：（1）在具有 16-18 个体节对的胚胎体节中，Pgk1 和 Actb 的组合提供了最佳的归一化效果，而 Actb 在更早的发育阶段也表现出高稳定性水平；（2）Gapdh 是在测试的两个发育阶段中前脑最稳定的基因；（3）在心脏样本中，Sdha、Gapdh 和 Actb 是 qPCR 归一化的最佳组合。对这三种组织的同时分析表明，Gapdh、Actb 和 Tbp 的组合是最可靠的内参。这项研究强调了根据细胞类型和/或感兴趣的组织选择合适的参考基因的重要性。此处描述的数据可应用于未来使用小鼠胚胎作为哺乳动物发育模型的研究。
