Anderson Kelli C, Elizur Abigail
Faculty of Science, Health and Education, University of the Sunshine Coast, Locked Bag 4, Maroochydore DC, Queensland 4558, Australia.
BMC Res Notes. 2012 Jan 10;5:21. doi: 10.1186/1756-0500-5-21.
The use of quantitative real-time polymerase chain reaction (qPCR) has become widespread due to its specificity, sensitivity and apparent ease of use. However, experimental error can be introduced at many stages during sample processing and analysis, and for this reason qPCR data are often normalised to an internal reference gene. The present study used three freely available algorithms (GeNorm, NormFinder and BestKeeper) to assess the stability of hepatically expressed candidate reference genes (Hprt1, Tbp, Ef1α and β-tubulin) in two experiments. In the first, female Atlantic salmon (Salmo salar) broodstock of different ages were reared at either 14 or 22 °C for an entire reproductive season, therefore a reference gene that does not respond to thermal challenge or reproductive condition was sought. In the second, estrogen treated juvenile salmon were maintained at the same temperatures for 14 days and a reference gene that does not respond to temperature or estrogen was required. Additionally, we performed independent statistic analysis to validate the outputs obtained from the program based analysis.
Based on the independent statistical analysis performed the stability of the genes tested was Tbp > Ef1α > Hprt1 > β-tubulin for the temperature/reproductive development experiment and Ef1α > Hprt1 > Tbp for the estrogen administration experiment (β-tubulin was not analysed). Results from the algorithms tested were quite ambiguous for both experiments; however all programs consistently identified the least stable candidate gene. BestKeeper provided rankings that were consistent with the independent analysis for both experiments. When an inappropriate candidate reference gene was used to normalise the expression of a hepatically expressed target gene, the ability to detect treatment-dependent changes in target gene expression was lost for multiple groups in both experiments.
We have highlighted the need to independently validate the results of reference gene selection programs. In addition, we have provided a reference point for those wishing to study the effects of thermal challenge and/or hormonal treatment on gene stability in Atlantic salmon and other teleost species.
定量实时聚合酶链反应（qPCR）因其特异性、敏感性及明显的易用性而得到广泛应用。然而，在样品处理和分析的多个阶段都可能引入实验误差，因此qPCR数据常被标准化为一个内参基因。本研究使用三种免费可用的算法（GeNorm、NormFinder和BestKeeper），在两个实验中评估肝脏表达的候选参考基因（Hprt1、Tbp、Ef1α和β-微管蛋白）的稳定性。在第一个实验中，不同年龄的雌性大西洋鲑（Salmo salar）亲鱼在14或22℃饲养整个繁殖季节，因此需要一个对热应激或繁殖条件无反应的参考基因。在第二个实验中，经雌激素处理的幼鲑在相同温度下饲养14天，需要一个对温度或雌激素无反应的参考基因。此外，我们进行了独立的统计分析，以验证基于程序分析获得的结果。
基于所进行的独立统计分析，在温度/生殖发育实验中，测试基因的稳定性为Tbp > Ef1α > Hprt1 > β-微管蛋白，在雌激素给药实验中为Ef1α > Hprt1 > Tbp（未分析β-微管蛋白）。两个实验中，所测试算法的结果都相当模糊；然而，所有程序都一致地识别出最不稳定的候选基因。BestKeeper提供的排名与两个实验的独立分析结果一致。当使用不合适的候选参考基因来标准化肝脏表达的靶基因的表达时，在两个实验的多个组中都失去了检测靶基因表达中依赖处理的变化的能力。
我们强调了独立验证参考基因选择程序结果的必要性。此外，我们为那些希望研究热应激和/或激素处理对大西洋鲑及其他硬骨鱼物种基因稳定性影响的人提供了一个参考点。
