The validity of a reference gene is highly dependent on the experimental conditions in green alga Ulva linza.

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.