Validation of Reference Genes for Quantitative Gene Expression Studies in Octopus minor

Widely distributed on tidal flats on the west and south coast of Korea, the long arm octopus Octopus minor is one of the important shellfish resources, as well as a key species in the siltymud tidal ecosystem. While several molecular studies have studied the genomes or the molecular phylogenetic relationship of O. minor with other cephalopod members, no studies to date have reported on gene expression in the long arm octopus. Reverse transcription quantitative polymerase chain reaction (RT-qPCR) is a commonly used method in gene expression studies; however, the accuracy and reliability of the assay results depend strongly on selection of appropriate reference genes. In this study, we first evaluated stabilities of 8 reference genes (elongation factor 1-beta, elongation factor 2, ribosomal protein, TATA-binding protein-associated factor 172, histone H4, elongation factor-gamma, succinate-hydroxymethylglutarate CoA-transferase, and transferrin) of O. minor distributed in the different tissues using geNorm. Our results indicated that TF was the most stable of the selected reference genes from the various tissues. To illustrate the usefulness of the new reference genes, expression analysis of heat shock protein70 related to environmental stresses was undertaken. Accordingly, this study provides suitable reference genes for RT-qPCR analysis that will enable future studies of gene expression in O. minor and other related species in cephalopods in order to gain a better understanding of their molecular responses to environmental stresses.