ACTB has been used previously for half-smooth tongue sole organs, but it was proved to be an unstable reference gene in our investigation because of significant difference between ACTB and the combination of GAPDH and 18S, even either of 18S or GAPDH. ACTB was ranked at the worst reference gene in tissues group. In contrast, ACTB/UBCE were shown to be the best reference genes in different organs of the Japanese flounder, and EF1A and RPSD were identified for Atlantic halibut and turbot tissue. We believe that this is the first work to assess valid reference genes for qRT-PCR studies in the half-smooth tongue sole. This work is important for future developmental gene expression studies in this commercially important species as it provides valuable tools for investigating gene expression in both the embryonic and larval stages, as well as in different tissues and following chemical treatment in this flatfish species, which currently suffers from a high mortality risk during larval production. Starch is the major storage carbohydrate in plants and an important renewable resource for both the food and non-food industry sectors. Starch is comprised of two glucose polymers and accumulates in plant tissues as semi-crystalline granules. NSC 136476 Amylopectin accounts for the majority of the granule mass. On average, there is one branch point every 20–25 glucose residues. However, the arrangement of branch points is thought to be non-random, such that linear chain segments can align together to form double helices that pack into stable, semi-crystalline lamellae. The branch points are concentrated in the amorphous regions between these crystalline lamellae. The higher-order structures adopted by amylopectin are thought to occur in all wild-type starches and underlie the water-insoluble granular characteristics of starch. Nevertheless, there is considerable variation in starch granule morphology, structure and composition between plant sources. These factors are important due to the impact they have on starch properties, which are relevant for downstream functional applications. A detailed understanding of how different biosynthetic enzymes influence amylopectin structure has been hindered by the fact that it is currently not possible to determine the exact structure of amylopectin. Also, we can only partially relate the physical properties of starch to its structure, and hence it is difficult to predictably control these characteristics through manipulation of enzyme abundance and/or specificities. Starch synthesis is mediated by three enzyme classes. Starch synthases extend a-1,4-linked chains by transferring new glucose units from ADP-glucose to the non-reducing end. Branch points are introduced by branching enzymes, which cleave an existing a1,4-bond of a linear chain and transfer the cut end to another chain, creating an a-1,6-bond. Both starch synthases and branching enzymes exist as multiple isoforms, thought to have different specificities.