Quantitative real-time polymerase chain reaction is a widely used technique that relies on reference genes for the normalisation of gene expression. These reference genes are constitutively expressed and must remain stable across all samples and treatments. Stability of housekeeping genes may vary and must be optimised for a specific tissue, sample or cell line. Here we present a study screening for possible reference gene candidates, eef1a1, rpl8, sub1.L, clta, H4 and odc1, in the Xenopus laevis (A6) kidney cell line. Quantification cycle results were analysed using geNorm to calculate the average expression stability and the coefficient of variation (CV) for each candidate reference gene. All of the tested genes met the guidelines for stable reference genes, namely an average expression stability of < 0.5 and a CV value of < 0.2, with eef1a1 > sub1.L > rpl8 > clta > odc1 > H4. By using pairwise variation analysis, the optimal number of reference targets was determined to be 2. As such, we report that the reference genes eef1a1 and sub1.L should be used to achieve optimal normalisation in A6 cells.

中文翻译：

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两栖动物肾源性 A6 上皮细胞定量实时 PCR 研究的参考基因验证

定量实时聚合酶链反应是一种广泛使用的技术，它依赖于参考基因进行基因表达的标准化。这些参考基因是组成型表达的，并且必须在所有样品和处理中保持稳定。管家基因的稳定性可能会有所不同，必须针对特定组织、样本或细胞系进行优化。在这里，我们在非洲爪蟾 (A6) 肾细胞系中对可能的参考基因候选者 eef1a1、rpl8、sub1.L、clta、H4 和 odc1 进行了研究筛选。使用 geNorm 分析定量循环结果以计算每个候选参考基因的平均表达稳定性和变异系数 (CV)。所有测试基因均符合稳定参考基因的指导方针，即平均表达稳定性<0.5和CV值<0.2，eef1a1 > sub1.L > rpl8 > clta > odc1 > H4。通过使用成对变异分析，参考目标的最佳数量被确定为 2。因此，我们报告应该使用参考基因​​ eef1a1 和 sub1.L 在 A6 细胞中实现最佳标准化。