Evaluation and validation of reference genes for qPCR analysis to study climate change-induced stresses in Sinularia cf. cruciata (Octocorallia: Alcyonidae)

• Seven housekeeping genes were tested for expression stability during two abiotic stresses.
• Thermal and pH stress-specific sets of HKGs are proposed as RGs for qPCR in Sinularia.
• ACTB, RPL12, SRP54 and TUBB genes were found most stable in a stress-dependent manner.
• TUBA and EF1A were found unsuitable for use as reference genes.
• Stress-dependent differential expression of HSP70 gene was detected.

Coral reef organisms, including octocorals, are facing the consequences of anthropogenic activities, such as increasing oceanic pH and sea surface temperature, threatening their long-term survival and well-being. Gene expression studies based on quantitative PCR (qPCR) are important tools to provide insight into the molecular basis of octocoral stress responses and their potential resilience mechanisms. Nevertheless, a lack of experimentally validated, stably expressed reference genes for the normalization of gene expression using quantitative reverse transcriptase PCR (qPCR) method limit such investigations among octocorals. Here, assessment of the expression stability of seven candidate reference genes was performed using a palette of statistical tools for valid qPCR-based gene expression studies on the octocoral Sinularia cf. cruciata during thermal (34 °C) and low-pH (pH 7.5) stress and the most suitable set of reference genes was determined for such experiments. The reliability of the selected reference genes was confirmed in a qPCR assay that targeted the heat shock protein 70 (HSP70) gene. The HSP70 gene was found to be significantly upregulated during thermal stress, whereas during low-pH stress the expression level of this gene decreased. This study provides experimentally validated stress-specific sets of stably expressed reference genes during climate change-induced stresses, which will benefit future gene expression studies on Sinularia cf. cruciata as well as other octocorals. These results also highlight potentially different acclimation strategies of octocorals to different sources of abiotic stresses, contributing to our understanding of the potential for the adaptation of coral reef organisms to a changing world.