Foquet Bert, Song Hojun
Department of Entomology, Texas A&M University, College Station, TX, United States of America.
PeerJ. 2020 Aug 3;8:e9618. doi: 10.7717/peerj.9618. eCollection 2020.
Reverse Transcriptase quantitative Polymerase Chain Reaction (RT-qPCR) is the current gold standard tool for the study of gene expression. This technique is highly dependent on the validation of reference genes, which exhibit stable expression levels among experimental conditions. Often, reference genes are assumed to be stable a priori without a rigorous test of gene stability. However, such an oversight can easily lead to misinterpreting expression levels of target genes if the references genes are in fact not stable across experimental conditions. Even though most gene expression studies focus on just one species, comparative studies of gene expression among closely related species can be very informative from an evolutionary perspective. In our study, we have attempted to find stable reference genes for four closely related species of grasshoppers (Orthoptera: Acrididae) that together exhibit a spectrum of density-dependent phenotypic plasticity. Gene stability was assessed for eight reference genes in two tissues, two experimental conditions and all four species. We observed clear differences in the stability ranking of these reference genes, both between tissues and between species. Additionally, the choice of reference genes clearly influenced the results of a gene expression experiment. We offer suggestions for the use of reference genes in further studies using these four species, which should be taken as a cautionary tale for future studies involving RT-qPCR in a comparative framework.
逆转录定量聚合酶链反应（RT-qPCR）是目前用于基因表达研究的金标准工具。该技术高度依赖于内参基因的验证，内参基因在实验条件下表现出稳定的表达水平。通常，内参基因在未经严格的基因稳定性测试的情况下就被先验地假定为稳定的。然而，如果内参基因在不同实验条件下实际上并不稳定，那么这种疏忽很容易导致对目标基因表达水平的错误解读。尽管大多数基因表达研究只关注一个物种，但从进化的角度来看，对近缘物种之间的基因表达进行比较研究可能会提供很多信息。在我们的研究中，我们试图为四种近缘蝗虫（直翅目：蝗科）找到稳定的内参基因，这四种蝗虫共同表现出一系列密度依赖性表型可塑性。在两种组织、两种实验条件以及所有四个物种中对八个内参基因的稳定性进行了评估。我们观察到这些内参基因在组织之间和物种之间的稳定性排名存在明显差异。此外，内参基因的选择明显影响了基因表达实验的结果。我们为在使用这四个物种的进一步研究中使用内参基因提供了建议，这应该作为在比较框架中涉及RT-qPCR的未来研究的一个警示故事。
