title: Validation of reference genes for cryopreservation studies with the gorgonian coral endosymbiont Symbiodinium.
abstract: Quantification by real-time RT-PCR requires a stable internal reference known as a housekeeping gene (HKG) for normalising the mRNA levels of target genes. The present study identified and validated stably expressed HKGs in post-thaw Symbiodinium clade G. Six potential HKGs, namely, pcna, gapdh, 18S rRNA, hsp90, rbcl, and ps1, were analysed using three different algorithms, namely, GeNorm, NormFinder, and BestKeeper. The GeNorm algorithm ranked the candidate genes as follows in the order of decreasing stability: pcna and gapdh > ps1 > 18S rRNA > hsp90 > rbcl. Results obtained using the NormFinder algorithm also showed that pcna was the most stable HKG and ps1 was the second most stable HKG. We found that the candidate HKGs examined in this study showed variable stability with respect to the three algorithms. These results indicated that both pcna and ps1 were suitable for normalising target gene expression determined by performing real-time RT-PCR in cryopreservation studies on Symbiodinium clade G. The results of the present study would help future studies to elucidate the effect of cryopreservation on gene expression in dinoflagellates.
authors.affiliation: Institute of Marine Biology, National Dong Hwa University, 2 Houwan Road, Checheng, Pingtung, 944, Taiwan.
authors.forename: Gabriella
authors.initials: G
authors.lastname: Chong
authors.affiliation: National museum of Marine Biology &Aquarium, 2 Houwan Road, Checheng, Pingtung, 944, Taiwan.
authors.forename: Fu-Wen
authors.initials: FW
authors.lastname: Kuo
authors.affiliation: Department of Biotechnology, Mingdao University, 369 Wen-Hua Road, Peetow, ChangHua, 52345, Taiwan.|Department of Post Modern Agriculture, Mingdao University, 369 Wen-Hua Road, Peetow, Chang Hua, 52345, Taiwan.
authors.forename: Sujune
authors.initials: S
authors.lastname: Tsai
authors.affiliation: Institute of Marine Biology, National Dong Hwa University, 2 Houwan Road, Checheng, Pingtung, 944, Taiwan.|National museum of Marine Biology &Aquarium, 2 Houwan Road, Checheng, Pingtung, 944, Taiwan.
authors.forename: Chiahsin
authors.initials: C
authors.lastname: Lin
mesh_terms: D000818:Animals; D037421:Anthozoa / Q000469:parasitology*; D015925:Cryopreservation / Q000379:methods*; D004141:Dinoflagellida / Q000235:genetics* / Q000502:physiology*; D020043:Genes, Essential; D060888:Real-Time Polymerase Chain Reaction / Q000379:methods* / Q000592:standards*; D012015:Reference Standards; D013559:Symbiosis