ABSTRACT 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.

中文翻译：

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不同培养条件下候选参考基因的基因表达稳定性定量PCR在金银藻中的Chattonella marina

摘要 选择合适的参考基因对于定量 PCR (qPCR) 中的相对定量很重要。我们研究了在不同辐照度、温度和氧化应激条件下以及不同生长阶段的金银藻属植物中 qPCR 的候选参考基因的表达稳定性。选择了九个候选参考基因（18S rRNA、细胞色素 c 氧化酶亚基 2、甘油醛-3-磷酸脱氢酶、肌动蛋白、α-微管蛋白、β-微管蛋白、钙调蛋白、60S 核糖体蛋白 L18 和延伸因子）并用于 qPCR 分析。qPCR 分析后，使用四种常用算法（geNorm、NormFinder、BestKeeper 和 ΔCt）评估基因表达稳定性。基于统计分析的综合评估，揭示钙调蛋白在所有条件和生长阶段都一直排在前三名。在不同温度（10、20 和 30 °C）下生长的 C. marina 中分析了过氧化物氧还蛋白（一种已知的抗氧化酶）的基因表达谱，以确认高级参考基因对相对定量的适用性。使用排名前三的基因（18S rRNA、α-微管蛋白和钙调蛋白）和排名第二的基因（伸长因子）的 2-Cys 过氧化物酶基因表达谱显示表达水平随温度而增加。然而，在三个温度下，排名最低的基因细胞色素 c 氧化酶亚基 2 没有显着差异。该结果表明，使用四种算法对候选参考基因的评估是有效的，