Selection of reference genes for quantitative real-time PCR normalization in Panax ginseng at different stages of growth and in different organs.
Selection of reference genes for quantitative real-time PCR normalization in Panax ginseng at different stages of growth and in different organs.
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DOI：
10.1371/journal.pone.0112177
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发表时间：
2014
期刊：
影响因子：
3.7
通讯作者：
Zhao Y
中科院分区：
文献类型：
--
作者：
Liu J;Wang Q;Sun M;Zhu L;Yang M;Zhao Y
定量实时反转录PCR （qRT-PCR）已成为广泛应用的基因表达分析方法；然而，其数据解释在很大程度上取决于内参基因的稳定性。人参是中药中最受欢迎的传统成分之一，其转录组学的研究越来越多。此外，利用qRT-PCR技术评估人参基因表达谱时，建立一系列可靠的内参基因是至关重要的。在本研究中，我们利用高通量测序平台生成的基因表达数据筛选人参候选内参基因。经统计检验，共筛选出20个内参基因（10个传统内参基因和10个新内参基因）。利用qPCR技术检测了这些基因在人参5个生长期和3个不同器官中的表达水平。随后使用geNorm、NormFinder和BestKeeper计算程序根据其表达的稳定性对这些基因进行排序和比较。虽然不同样品的最佳内参基因存在差异，但CYP和EF-1α是所有样品中最稳定的基因。GAPDH/30S RPS20、CYP/60S RPL13和CYP/QCR是根、茎、叶的最佳内参基因对。CYP/60S RPL13、CYP/eIF-5A、aTUB/V-ATP、eIF-5A/SAR1和aTUB/pol IIa是五个发育阶段中表达最稳定的组合。本研究为建立准确的qRT-PCR方法奠定了基础，并为今后人参基因表达谱的研究奠定了基础。
Quantitative real-time reverse transcription PCR (qRT-PCR) has become a widely used method for gene expression analysis; however, its data interpretation largely depends on the stability of reference genes. The transcriptomics of Panax ginseng, one of the most popular and traditional ingredients used in Chinese medicines, is increasingly being studied. Furthermore, it is vital to establish a series of reliable reference genes when qRT-PCR is used to assess the gene expression profile of ginseng. In this study, we screened out candidate reference genes for ginseng using gene expression data generated by a high-throughput sequencing platform. Based on the statistical tests, 20 reference genes (10 traditional housekeeping genes and 10 novel genes) were selected. These genes were tested for the normalization of expression levels in five growth stages and three distinct plant organs of ginseng by qPCR. These genes were subsequently ranked and compared according to the stability of their expressions using geNorm, NormFinder, and BestKeeper computational programs. Although the best reference genes were found to vary across different samples, CYP and EF-1α were the most stable genes amongst all samples. GAPDH/30S RPS20, CYP/60S RPL13 and CYP/QCR were the optimum pair of reference genes in the roots, stems, and leaves. CYP/60S RPL13, CYP/eIF-5A, aTUB/V-ATP, eIF-5A/SAR1, and aTUB/pol IIa were the most stably expressed combinations in each of the five developmental stages. Our study serves as a foundation for developing an accurate method of qRT-PCR and will benefit future studies on gene expression profiles of Panax Ginseng.
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影响因子：
1.5
作者：
Kiselev, K. V.;Grishchenko, O. V.;Zhuravlev, Y. N.
通讯作者：
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