Piazza Verónica G, Bartke Andrzej, Miquet Johanna G, Sotelo Ana I
Universidad de Buenos Aires, Consejo Nacional de Investigaciones Científicas y Técnicas, Instituto de Química y Fisicoquímica Biológicas (IQUIFIB), Facultad de Farmacia y Bioquímica, Buenos Aires C1113AAD, Argentina.
Department of Internal Medicine and Physiology, Division of Geriatric Research, School of Medicine, Southern Illinois University, Springfield, IL 62794-9628, USA.
Int J Mol Sci. 2017 May 16;18(5):1060. doi: 10.3390/ijms18051060.
The reliability of reverse transcription-quantitative PCR (RT-qPCR) results in gene expression studies depends on the approaches used to account for non-biological variations. In order to find a proper normalization strategy for the study of genes related to growth hormone signaling in skeletal muscle of growing mice, nine unrelated genes were evaluated as internal controls. According to the most used algorithms-geNorm, the Comparative Δq method, NormFinder and BestKeeper-, , and were found as the most stable. However, the relative expression levels of eight of the potential reference genes assessed decreased with age in cDNA samples obtained from the same amount of total RNA. In a different approach to analyze this apparent discrepancy, experiments were performed with cDNA obtained from equal amounts of purified mRNA. Since the decline was still observed, the hypothesis of an age-related change in mRNA to total RNA ratio that could account for the systematic decrease was rejected. Differences among experimental groups could be due to a substantial increase with age in highly expressed mRNAs, which would bias the quantitation of the remaining genes. Consequently, those reference genes reflecting this dilution effect, which would have been discarded considering their variable relative expression levels, arose as suitable internal controls.
逆转录定量聚合酶链反应（RT-qPCR）结果在基因表达研究中的可靠性取决于用于处理非生物学变异的方法。为了找到一种合适的标准化策略来研究生长中小鼠骨骼肌中与生长激素信号相关的基因，评估了九个不相关基因作为内参。根据最常用的算法——geNorm、比较Δq法、NormFinder和BestKeeper——发现 、 和 是最稳定的。然而，在从相同总量的总RNA获得的cDNA样本中，所评估的八个潜在参考基因的相对表达水平随年龄下降。在分析这种明显差异的另一种方法中，使用从等量纯化mRNA获得的cDNA进行实验。由于仍然观察到下降，关于mRNA与总RNA比率随年龄变化可解释这种系统性下降的假设被否定。实验组之间的差异可能是由于高表达mRNA随年龄大幅增加，这会使其余基因的定量产生偏差。因此，那些反映这种稀释效应的参考基因，尽管考虑到它们可变的相对表达水平会被舍弃，但却成为了合适的内参。
