Reference gene selection for qRT-PCR assays in Stellera chamaejasme subjected to abiotic stresses and hormone treatments based on transcriptome datasets.
Reference gene selection for qRT-PCR assays in Stellera chamaejasme subjected to abiotic stresses and hormone treatments based on transcriptome datasets.
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基于转录组数据集，对经非生物胁迫和激素处理的狼毒进行 qRT-PCR 检测的参考基因选择
作者：
Liu X;Guan H;Song M;Fu Y;Han X;Lei M;Ren J;Guo B;He W;Wei Y
研究背景狼毒是我国重要的草原有毒植物，对人畜有毒害作用。S.的快速扩张。狼毒对草原生态造成极大破坏，严重危害畜牧业的发展。为了制定有效的防治措施，迫切需要从遗传水平上研究其在不同不利生境中的适应和扩展机制。定量实时聚合酶链反应（qRT-PCR）是一种广泛用于在转录水平研究基因表达的技术;然而，qRT-PCR需要参考基因（RG）作为数据标准化的内源性对照，只有通过适当的RG选择和qRT-PCR才能保证表达研究和RNA-seq数据分析的可靠性和稳健性。然而，目前对S.据报道，方法本研究从链球菌转录组数据库中筛选出10个候选RG，即18 S、60 S、CYP、GAPCP 1、GAPDH 2、EF 1B、MDH、SAND、TUA 1和TUA 6。用geNorm、Normandy和BestKeeper程序分析了它们在干旱、低温和盐胁迫下的表达稳定性以及在脱落酸（阿坝）、赤霉素（GA）、乙烯利（ETH）等激素处理下的表达稳定性。结果GAPCP 1和EF 1B是3种非生物胁迫下的最佳组合，TUA 6和SAND、TUA 1和NAR、GAPDH 2和60 S分别是阿坝、GA和ETH处理下的最佳组合。此外，GAPCP 1和60 S被评估为所有样品的最佳组合，18 S是用作所有实验子集中内部对照的最不稳定的RG。两个靶基因（P5 CS2和GI）的表达模式进一步验证了我们选择的RG适合于基因表达标准化。讨论本工作首次尝试对S.狼毒我们的研究结果为qRT-PCR分析中的高精度归一化提供了合适的RG，从而使在这些实验条件下分析基因表达更加方便。
Background Stellera chamaejasme Linn, an important poisonous plant of the China grassland, is toxic to humans and livestock. The rapid expansion of S. chamaejasme has greatly damaged the grassland ecology and, consequently, seriously endangered the development of animal husbandry. To draft efficient prevention and control measures, it has become more urgent to carry out research on its adaptive and expansion mechanisms in different unfavorable habitats at the genetic level. Quantitative real-time polymerase chain reaction (qRT-PCR) is a widely used technique for studying gene expression at the transcript level; however, qRT-PCR requires reference genes (RGs) as endogenous controls for data normalization and only through appropriate RG selection and qRT-PCR can we guarantee the reliability and robustness of expression studies and RNA-seq data analysis. Unfortunately, little research on the selection of RGs for gene expression data normalization in S. chamaejasme has been reported. Method In this study, 10 candidate RGs namely, 18S, 60S, CYP, GAPCP1, GAPDH2, EF1B, MDH, SAND, TUA1, and TUA6, were singled out from the transcriptome database of S. chamaejasme, and their expression stability under three abiotic stresses (drought, cold, and salt) and three hormone treatments (abscisic acid, ABA; gibberellin, GA; ethephon, ETH) were estimated with the programs geNorm, NormFinder, and BestKeeper. Result Our results showed that GAPCP1 and EF1B were the best combination for the three abiotic stresses, whereas TUA6 and SAND, TUA1 and CYP, GAPDH2 and 60S were the best choices for ABA, GA, and ETH treatment, respectively. Moreover, GAPCP1 and 60S were assessed to be the best combination for all samples, and 18S was the least stable RG for use as an internal control in all of the experimental subsets. The expression patterns of two target genes (P5CS2 and GI) further verified that the RGs that we selected were suitable for gene expression normalization. Discussion This work is the first attempt to comprehensively estimate the stability of RGs in S. chamaejasme. Our results provide suitable RGs for high-precision normalization in qRT-PCR analysis, thereby making it more convenient to analyze gene expression under these experimental conditions.
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