Qingdao Agricultural University, China.
Curr Genet. 2012 Feb;58(1):13-20. doi: 10.1007/s00294-011-0361-3. Epub 2011 Dec 29.
Normalization based on inappropriate reference gene may lead to the reduction of the accuracy of RT-qPCR. Although determination of suitable reference genes is essential to RT-qPCR studies, reports on the evaluation of reference genes in Ulva linza, a ubiquitous green-tide forming alga, are lacking. The expression levels of ten candidate reference genes were analyzed in U. linza across different experimental treatments, and the best-ranked reference genes differed across the treatments. The most suitable reference genes were tubulin2 (TUB2) among different salinity and UV treatments. Histone 2 (H2) was stably expressed in different temperature and desiccation stress treatments. 18S rRNA exhibited better expression stability in different light intensity treatments. While all tested samples were considered, none of single gene was widely applicable as a reference gene. Moreover, using a combination of two genes as reference genes might improve the reliability of gene expression by RT-qPCR, and the combination of TUB1 and TUB2 was selected as ideal for all tested samples. The results suggest that assessing the stability of reference gene expression patterns, determining candidates, and testing their suitability are required for each experimental investigation. The results will guide the selection of reference genes for gene expression studies in U. linza.
基于不合适的参照基因进行归一化可能会导致 RT-qPCR 准确性降低。尽管确定合适的参照基因对于 RT-qPCR 研究至关重要，但目前缺乏关于广泛分布的绿潮形成藻类石莼中参照基因评估的报告。本研究分析了石莼在不同实验处理下的 10 个候选参照基因的表达水平，不同处理下最佳参照基因的排名不同。在不同盐度和 UV 处理中，微管蛋白 2（TUB2）是最适合的参照基因。组蛋白 2（H2）在不同温度和干燥胁迫处理中稳定表达。在不同光照强度处理中，18S rRNA 的表达稳定性较好。考虑到所有测试样本，没有一个单一的基因可以作为广泛适用的参照基因。此外，使用两个基因的组合作为参照基因可能会提高 RT-qPCR 中基因表达的可靠性，并且选择 TUB1 和 TUB2 的组合作为所有测试样本的理想组合。结果表明，需要针对每个实验研究评估参照基因表达模式的稳定性、确定候选基因并测试其适用性。该结果将为石莼基因表达研究中参照基因的选择提供指导。
