ABSTRACT Quantitative real-time reverse-transcription polymerase chain reaction (qRT-PCR) is an effective and widely used method in gene expression studies. Selection of appropriate reference genes is key to generating reliable data using this technique and there are no published studies regarding the selection of reliable reference genes in the genus Primula. In this study, 10 candidate reference genes (28S, ACT, TUA, TUB, HIS, GAPDH, RPL18a, STPP, CSD and RNA pol II), were chosen from the transcriptome database in Primula forbesii Franch., and their expression levels were assessed by qRT-PCR in different flower developmental stages and different organs. Four software programs; geNorm, NormFinder, Bestkeeper, and Ref-Finder were used to validate the stability and suitability of potential reference genes. Comprehensive results showed that GAPDH was the most stable reference gene for P. forbesii, while TUA and TUB were the most unstable ones in flower development and different organs, respectively. Finally, to illustrate the usefulness of the top-ranked reference genes, the expression profile of the P. forbesii linalool synthase/nerolidol synthase gene (PfLIS/NES) was analysed, which highlighted the importance of proper reference gene selection. This work represents the first systematic study of reference gene stability in Primula and lays the foundation for future research into Primula gene expression patterns.

中文翻译：

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qRT-PCR在报春花发育过程中和不同器官中的参考基因选择和验证

摘要 定量实时逆转录聚合酶链反应 (qRT-PCR) 是基因表达研究中一种有效且广泛使用的方法。选择合适的参考基因是使用该技术生成可靠数据的关键，目前还没有关于在报春花属中选择可靠参考基因的已发表研究。本研究从 Primula forbesii Franch. 的转录组数据库中选取 10 个候选参考基因（28S、ACT、TUA、TUB、HIS、GAPDH、RPL18a、STPP、CSD 和 RNA pol II），并评估它们的表达水平通过 qRT-PCR 在不同的花发育阶段和不同的器官。四个软件程序；geNorm、NormFinder、Bestkeeper 和 Ref-Finder 用于验证潜在参考基因的稳定性和适用性。综合结果表明，GAPDH是福布斯最稳定的参考基因，而TUA和TUB分别是花发育和不同器官最不稳定的参考基因。最后，为了说明排名靠前的参考基因的有用性，我们分析了福布斯假单胞菌芳樟醇合酶/橙花醇合酶基因 (PfLIS/NES) 的表达谱，这突出了正确选择参考基因的重要性。这项工作代表了对报春花中参考基因稳定性的首次系统研究，并为未来对报春花基因表达模式的研究奠定了基础。分析了福布斯假单胞菌芳樟醇合酶/橙花醇合酶基因 (PfLIS/NES) 的表达谱，这突出了正确选择参考基因的重要性。这项工作代表了对报春花中参考基因稳定性的首次系统研究，并为未来对报春花基因表达模式的研究奠定了基础。分析了福布斯假单胞菌芳樟醇合酶/橙花醇合酶基因 (PfLIS/NES) 的表达谱，这突出了正确选择参考基因的重要性。这项工作代表了对报春花中参考基因稳定性的首次系统研究，并为未来对报春花基因表达模式的研究奠定了基础。