Institute of Marine Biology, National Dong Hwa University, 2 Houwan Road, Checheng, Pingtung, 944, Taiwan.
National museum of Marine Biology &Aquarium, 2 Houwan Road, Checheng, Pingtung, 944, Taiwan.
Sci Rep. 2017 Jan 9;7:39396. doi: 10.1038/srep39396.
Quantification by real-time RT-PCR requires a stable internal reference known as a housekeeping gene (HKG) for normalising the mRNA levels of target genes. The present study identified and validated stably expressed HKGs in post-thaw Symbiodinium clade G. Six potential HKGs, namely, pcna, gapdh, 18S rRNA, hsp90, rbcl, and ps1, were analysed using three different algorithms, namely, GeNorm, NormFinder, and BestKeeper. The GeNorm algorithm ranked the candidate genes as follows in the order of decreasing stability: pcna and gapdh > ps1 > 18S rRNA > hsp90 > rbcl. Results obtained using the NormFinder algorithm also showed that pcna was the most stable HKG and ps1 was the second most stable HKG. We found that the candidate HKGs examined in this study showed variable stability with respect to the three algorithms. These results indicated that both pcna and ps1 were suitable for normalising target gene expression determined by performing real-time RT-PCR in cryopreservation studies on Symbiodinium clade G. The results of the present study would help future studies to elucidate the effect of cryopreservation on gene expression in dinoflagellates.
实时 RT-PCR 定量需要一个稳定的内参，称为管家基因 (HKG)，用于标准化靶基因的 mRNA 水平。本研究鉴定并验证了解冻后的 Symbiodinium 类群 G 中稳定表达的 HKG。使用三种不同的算法，即 GeNorm、NormFinder 和 BestKeeper，分析了 6 个潜在的 HKG，即 pcna、gapdh、18S rRNA、hsp90、rbcl 和 ps1。GeNorm 算法按照稳定性降低的顺序将候选基因排列如下：pcna 和 gapdh>ps1>18S rRNA>hsp90>rbcl。NormFinder 算法的结果也表明 pcna 是最稳定的 HKG，ps1 是第二稳定的 HKG。我们发现，本研究中检查的候选 HKG 相对于这三种算法显示出不同的稳定性。这些结果表明，pcna 和 ps1 都适合用于对 Symbiodinium 类群 G 的冷冻保存研究中实时 RT-PCR 确定的靶基因表达进行归一化。本研究的结果将有助于未来的研究阐明冷冻保存对甲藻基因表达的影响。
