Department of Biology and Environmental Science, University of Sussex, Falmer, Brighton BN1 9QJ, UK.
Environ Sci Pollut Res Int. 2011 Aug;19(7):2728-33. doi: 10.1007/s11356-012-0772-9.
The aim of this study is to develop a normalization method for real-time PCR data by analyzing the most stably expressed control genes in mussel (Mytilus edulis) reproductive tissue.
To facilitate this, six candidate genes, including several commonly used in the literature, were investigated in mussels at different stages of gametogenesis and following experimental exposure to a model estrogen (17b-estradiol). GeNorm and NormFinder softwares were employed to assess the stability of the reference genes.
Our results demonstrate that the most stable reference genes are not the same in mussels at different stages of gametogenesis and in experimentally E2-exposed mussels. Interestingly, HEL (helicase) and ACT (actin) mRNA expression levels were most affected by the stage of gametogenesis and yet, in molluscan studies, ACT is possibly the most frequently used reference gene.
We demonstrate that the experimental results are highly dependent on the reference gene chosen and that statistically significant contrasting differences between sample groups are present or absent depending on the reference gene employed.
本研究旨在通过分析贻贝（Mytilus edulis）生殖组织中最稳定表达的控制基因，开发一种实时 PCR 数据的归一化方法。
为此，在贻贝配子发生的不同阶段以及暴露于模型雌激素（17β-雌二醇）后的实验中，研究了 6 个候选基因，包括文献中常用的几个基因。使用 GeNorm 和 NormFinder 软件评估参考基因的稳定性。
我们的结果表明，在贻贝配子发生的不同阶段和实验性 E2 暴露的贻贝中，最稳定的参考基因并不相同。有趣的是，HEL（解旋酶）和 ACT（肌动蛋白）mRNA 表达水平受配子发生阶段的影响最大，但在软体动物研究中，ACT 可能是最常用的参考基因。
我们证明实验结果高度依赖于所选的参考基因，并且根据所使用的参考基因，样本组之间存在或不存在统计学上显著的对比差异。
