Bower Neil I, Johnston Ian A
Scottish Oceans Institute, School of Biology, University of St Andrews, St Andrews, Fife, KY16 8LB, UK.
BMC Mol Biol. 2009 Aug 10;10:80. doi: 10.1186/1471-2199-10-80.
Relatively few studies have used cell culture systems to investigate gene expression and the regulation of myogenesis in fish. To produce robust data from quantitative real-time PCR mRNA levels need to be normalised using internal reference genes which have stable expression across all experimental samples. We have investigated the expression of eight candidate genes to identify suitable reference genes for use in primary myogenic cell cultures from Atlantic salmon (Salmo salar L.). The software analysis packages geNorm, Normfinder and Best keeper were used to rank genes according to their stability across 42 samples during the course of myogenic differentiation.
Initial results showed several of the candidate genes exhibited stable expression throughout myogenic culture while Sdha was identified as the least stable gene. Further analysis with geNorm, Normfinder and Bestkeeper identified Ef1alpha, Hprt1, Ppia and RNApolII as stably expressed. Comparison of data normalised with the geometric average obtained from combinations of any three of these genes showed no significant differences, indicating that any combination of these genes is valid.
The geometric average of any three of Hprt1, Ef1alpha, Ppia and RNApolII is suitable for normalisation of gene expression data in primary myogenic cultures from Atlantic salmon.
相对较少的研究使用细胞培养系统来研究鱼类中的基因表达和肌生成调控。为了从定量实时PCR获得可靠数据，需要使用在所有实验样本中表达稳定的内参基因对mRNA水平进行标准化。我们研究了八个候选基因的表达，以确定适合用于大西洋鲑（Salmo salar L.）原代肌细胞培养的内参基因。使用软件分析包geNorm、Normfinder和Best keeper根据它们在肌生成分化过程中42个样本中的稳定性对基因进行排名。
初步结果显示，几个候选基因在整个肌生成培养过程中表现出稳定表达，而Sdha被确定为最不稳定的基因。使用geNorm、Normfinder和Bestkeeper进行的进一步分析确定Ef1alpha、Hprt1、Ppia和RNApolII表达稳定。用这三个基因组合得到的几何平均值对数据进行标准化后的比较显示没有显著差异，表明这些基因的任何组合都是有效的。
Hprt1、Ef1alpha、Ppia和RNApolII中任意三个基因的几何平均值适用于标准化大西洋鲑原代肌细胞培养中的基因表达数据。
