Zhou Rui-Xue, Meng Tao, Meng Hai-Bo, Cheng Dun-Xue, Bin Shi-Yu, Cheng Jia, Fu Gui-Hong, Chu Wu-Ying, Zhang Jian-She
College of Biological Engineering and Environmental Sciences, Changsha University, Changsha 410003, China.
Dongwuxue Yanjiu. 2010 Apr;31(2):141-6. doi: 10.3724/SP.J.1141.2010.02141.
At present, transcription analysis of gene expression commonly uses housekeeping genes as control for normalization. In this study, the expression levels of three housekeeping genes including GAPDH, beta-actin, and 18S rRNA in six tissues and five developmental stages of the Mandarin fish Siniperca chuatsi were assayed with quantitative real-time PCR (qPCR). Differences in expression levels were analyzed using geNorm program. The results demonstrate that beta-actin is the most stable gene at developmental stages and GAPDH is the most stable in different tissues. While 18S rRNA expression during development is differentially regulated, which indicates it is suitable as an internal control for gene expression normalization at the developmental level. Overall, the data suggest that the two most stable housekeeping genes are enough to accurately calibrate gene expression in S. chuatsi. The significance of this study provided convincing references and methodology for housekeeping gene selection and normalization in gene expression analysis with regular PCR or qPCR.
目前，基因表达的转录分析通常使用管家基因作为标准化对照。在本研究中，采用定量实时PCR（qPCR）检测了鳜鱼（Siniperca chuatsi）六个组织和五个发育阶段中包括甘油醛-3-磷酸脱氢酶（GAPDH）、β-肌动蛋白和18S核糖体RNA（rRNA）在内的三个管家基因的表达水平。使用geNorm程序分析表达水平的差异。结果表明，β-肌动蛋白在发育阶段是最稳定的基因，而GAPDH在不同组织中最稳定。虽然18S rRNA在发育过程中的表达受到差异调节，这表明它适合作为发育水平上基因表达标准化的内参。总体而言，数据表明两个最稳定的管家基因足以准确校准鳜鱼中的基因表达。本研究的意义为常规PCR或qPCR基因表达分析中管家基因的选择和标准化提供了有说服力的参考和方法。
