Potential utility of 14 candidate housekeeping genes as normalization reference for RT-qPCR analysis with developmental samples (fertilized eggs to late veliger larvae) in Pacific abalone Haliotis discus hannai was evaluated using four different statistical algorithms (geNorm, NormFinder, BestKeeper and comparative ΔCT method). Different algorithms identified different genes as the best candidates, and geometric mean-based final ranking from the most to the least stable expression was as follow: RPL5, RPL4, RPS18, RPL8, RPL7, UBE2, RPL7A, GAPDH, RPL36, PPIB, EF1A, ACTB and B-TU. The findings were further validated via relative quantification of metallothionein (MT) transcripts using the stable and unstable reference genes, and expression levels of MT were greatly influenced according to the choice of reference genes. In overall, our data suggest that RPL5 and RPS18, either singly or in combination, are appropriate for normalizing gene expression in developmental samples of this abalone species, whereas ACTB, B-TU and EF1A are less stable and not recommended. In addition, our findings propose that standard deviations in geometric ranking as well as geometric mean itself should also be taken into account for the final selection of reference gene(s). This study could be a useful basis to facilitate the generation of accurate and reliable RT-qPCR data with developmental samples in this abalone species.

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
CONFLICTS OF INTEREST
ACKNOWLEDGEMENTS
AUTHOR'S AFFILIATION, POSITION ANDORCID NO.
REFERENCES

• Sang Yoon Lee(Department of Marine Bio-Materials and Aquaculture, Pukyong National University, Busan 48513, Korea)
• Choul-Ji Park(Genetics and Breeding Research Center, National Institute of Fisheries Science, Geoje 53334, Korea)
• Yoon Kwon Nam(Department of Marine Bio-Materials and Aquaculture, Pukyong National University, Busan 48513, Korea) Corresponding author