Key Laboratory of Plant Secondary Metabolism and Regulation of Zhejiang Province, College of Life Science and Medicine, Zhejiang Sci-Tech University, Hangzhou 310018, China.
Tasly Botanical Pharmaceutical Co., Ltd., Shangluo 726000, China.
Genes (Basel). 2022 Oct 25;13(11):1947. doi: 10.3390/genes13111947.
The quantitative real-time PCR (qRT-PCR) is an efficient and sensitive method for determining gene expression levels, but the accuracy of the results substantially depends on the stability of the reference gene (RG). Therefore, choosing an appropriate reference gene is a critical step in normalizing qRT-PCR data. L. is a traditional Chinese medicine herb widely used in China. Its main medicinal part is the fruiting spike which is termed Spica Prunellae. However, thus far, few studies have been conducted on the mechanism of Spica Prunellae development. Meanwhile, no reliable RGs have been reported in The expression levels of 14 candidate RGs were analyzed in this study in various organs and at different stages of Spica Prunellae development. Four statistical algorithms (Delta Ct, BestKeeper, NormFinder, and geNorm) were utilized to identify the RGs' stability, and an integrated stability rating was generated via the RefFinder website online. The final ranking results revealed that was the most stable RG, whereas was the least suitable as an RG. Furthermore, + was identified as the best RG combination in different periods and the total samples. Finally, the expressions of the and genes related to the regulation of rosmarinic acid synthesis in different organs were used to verify the stable and unstable RGs. The stable RGs in were originally identified and verified in this work. This achievement provides strong support for obtaining a reliable qPCR analysis and lays the foundation for in-depth research on the developmental mechanism of Spica Prunellae.
实时荧光定量 PCR（qRT-PCR）是一种测定基因表达水平的高效、敏感的方法，但结果的准确性在很大程度上取决于参照基因（RG）的稳定性。因此，选择合适的参照基因是对 qRT-PCR 数据进行标准化的关键步骤。L. 是一种在中国广泛使用的中药，其主要药用部位是被称为夏枯草的果穗。然而，迄今为止，对夏枯草发育的机制研究甚少。同时，尚未报道在夏枯草发育的不同阶段和不同器官中稳定表达的 RG。本研究分析了 14 个候选 RG 在不同器官和不同发育阶段的表达水平。利用 4 种统计算法（Delta Ct、BestKeeper、NormFinder 和 geNorm）对 RG 的稳定性进行鉴定，并通过 RefFinder 网站在线生成综合稳定性评分。最终的排序结果表明，是最稳定的 RG，而则最不适合作为 RG。此外，在不同时期和总样本中，+被确定为最佳 RG 组合。最后，使用不同器官中与迷迭香酸合成调控相关的和基因的表达来验证稳定和不稳定的 RG。本研究在不同器官中鉴定和验证了稳定的 RG。这一成就为获得可靠的 qPCR 分析提供了有力支持，并为深入研究夏枯草的发育机制奠定了基础。
