Gene expression stability of candidate reference genes under different culture conditions for quantitative PCR in the raphidophyte Chattonella marina

Selection of suitable reference genes is important for relative quantification in quantitative PCR (qPCR). We investigated the expression stability of candidate reference genes for qPCR in the raphidophyte Chattonella marina at different irradiance, temperature, and oxidative stress conditions, and in different growth phases. Nine candidate reference genes (18S rRNA, cytochrome c oxidase subunit 2, glyceraldehyde-3-phosphate dehydrogenase, actin, alpha-tubulin, beta-tubulin, calmodulin, 60S ribosomal protein L18, and elongation factor) were selected and used for qPCR analysis. After qPCR analysis, gene expression stabilities were evaluated using four frequently used algorithms (geNorm, NormFinder, BestKeeper and ΔCt). A comprehensive evaluation, based on statistical analysis, revealed that calmodulin constantly ranked in the top three at all conditions and growth phases. The gene expression profile of peroxiredoxin, a known antioxidant enzyme, was analysed in C. marina grown at different temperatures (10, 20, and 30 °C) to confirm the applicability of the high-ranked reference genes to relative quantification. The 2-Cys peroxiredoxin gene expression profile using the top-three ranked genes (18S rRNA, alpha-tubulin, and calmodulin) and the second-lowest ranked gene (elongation factor) showed a temperature-dependent increase in expression levels. However, there was no significant difference in the lowest ranked gene, cytochrome c oxidase subunit 2, at the three temperatures. This result showed that evaluation of the candidate reference genes using the four algorithms was valid, and indicates the importance of reference gene selection for relative qPCR in C. marina.