Types and following treatment with SB-431542, an inhibitor of the Nodal signaling pathway. Three statistical algorithms, geNorm, BestKeeper and NormFinder, were used to analyze the qRT-PCR data. In addition, the ontogenesis expression profiles of LEFTY were investigated to confirm the efficacy of the selected reference genes as this is reported to be part of the Nodal-pathway that plays a role in controlling eye laterality in the flounder, as well as being shown to be expressed on the left side of the dorsal diencephalon and internal organs to form fixed laterality. The results obtained in this study provide an essential tool for future gene expression studies in the half-smooth tongue sole. The qRT-PCR method is widely used in gene expression studies of both freshwater and marine fish, when investigating a variety of conditions, such as infection and stress, as well as in various cell lines and different tissues. As a crucial step, the qRT-PCR data should be accurately normalized by use of the appropriate reference genes. Otherwise, the use of non-validated reference genes can lead to erroneous conclusions that are biologically meaningless. However, there is no universally applicable reference gene. The same reference genes in different species and different reference genes in the same species can have dramatically different expression levels under diverse experimental conditions. For example, GAPDH has been validated as the most stable reference gene in the mandarin fish, but has significant expression variation in the Atlantic halibut and zebrafish. On the other hand, ACTB and Tubb2 showed less expression variation, but 18S rRNA and GAPDH varied significantly, even these reference genes in the same species, Atlantic halibut. Thus, to improve the sensitivity and reliability of qRT-PCR in practice, the validation of reference genes should be performed for each different species, tissue type and experiment condition to be used. The results obtained in our study were analyzed with geNorm, BestKeeper and NormFinder. The most stable reference genes Z-VAD-FMK generated from these three software applications showed a high level of similarity; however there were subtle deviations in the rank order. For example, throughout all developmental stages, the top 3 stable expression genes were: GAPDH, B2M and 18S for geNorm; GAPDH, 18S and B2M for Bestkeeper; and 18S, GAPDH and UBCE for NormFinder. This deviation is natural because of the different algorithms used. Both BestKeeper and geNorm use a pair-wise comparison approach, and are highly dependent on the assumption that none of the genes being analyzed are co-regulated. On the other hand, in NormFinder, a separate analysis of the sample subgroups and estimation of both intra- and inter-group variation in expression levels are included into the calculation of a gene stability value. This is consistent with the results reported in the Atlantic salmon and Japanese flounder. In addition, tissuedependent variations have been observed in the expression of most housekeeping genes in flatfish.