Institute of Evolution & Marine Biodiversity, Ocean University of China, Qingdao 266003, China.
Fish Shellfish Immunol. 2012 Jun;32(6):1068-73. doi: 10.1016/j.fsi.2012.03.001. Epub 2012 Mar 12.
Amphioxus is a well-known model organism widely used for interspecies comparative genome study, developmental homology analysis and comparative immunological investigation. However, no study has been performed so far to evaluate the internal reference for quantitative RT-PCR (qRT-PCR) studies of gene expression in this important species. In this study, two software applications (geNorm and NormFinder) were used to evaluate the expression stability of 4 housekeeping genes (ACTB, GAPDH, 18S rRNA and EF1α) in 8 different normal tissues (whole body, gut, gut-free body, hepatic caecum, gill, hind-gut, notochord and muscle) and 2 tissues (gut and gut-free body) challenged with LPS and LTA in amphioxus Branchiostoma japonicum. Our results showed that in the normal tissues, the expression of 18S rRNA was most abundant, whereas the expression levels of the other three genes were close to each other, with the expression of ACTB being most unstable. Following challenge with LPS and LTA, all the four genes exhibited varied degrees of expression changes in the different tissues and the expression stabilities of the genes were also affected by the different experimental conditions. Yet, the overall ranking results produced by the two algorithms consistently indicated that the expression of EF1α showed the most least variation in the different tissues, suggesting that EF1α is a suitable internal control for qRT-PCR studies in amphioxus B. japonicum.
文昌鱼是一种广为人知的模式生物，广泛用于种间比较基因组研究、发育同源性分析和比较免疫学研究。然而，迄今为止，尚无研究评估该重要物种基因表达定量 RT-PCR(qRT-PCR)研究的内参。在这项研究中，使用了两种软件应用程序（geNorm 和 NormFinder）来评估 4 种管家基因（ACTB、GAPDH、18S rRNA 和 EF1α）在 8 种不同正常组织（整体、肠道、无肠道体、肝盲囊、鳃、后肠、脊索和肌肉）和 2 种组织（肠道和无肠道体）中的表达稳定性，这两种组织受到 LPS 和 LTA 的刺激。我们的结果表明，在正常组织中，18S rRNA 的表达最丰富，而其他三个基因的表达水平彼此接近，其中 ACTB 的表达最不稳定。在受到 LPS 和 LTA 刺激后，所有 4 个基因在不同组织中表现出不同程度的表达变化，基因的表达稳定性也受到不同实验条件的影响。然而，两种算法产生的总体排名结果一致表明，EF1α 在不同组织中的表达变化最小，表明 EF1α 是文昌鱼 B. japonicum qRT-PCR 研究的合适内参。
