Selection of reference genes for gene expression normalization in Pyropia yezoensis using quantitative real-time PCR
Selection of reference genes for gene expression normalization in Pyropia yezoensis using quantitative real-time PCR
复制标题
使用定量实时 PCR 选择条斑紫菜基因表达标准化的内参基因
DOI：
10.1007/s10811-014-0359-6
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发表时间：
2015-04-01
影响因子：
3.3
通讯作者：
Mao, Yunxiang
中科院分区：
文献类型：
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作者：
Kong, Fanna;Cao, Min;Mao, Yunxiang
实时荧光定量PCR （Quantitative real-time PCR, qRT-PCR）是常用的基因表达分析方法。为了使变异归一化，需要选择内参基因，以避免对实验结果的误读和错误分析。叶藻（Pyropia yezoensis）是一种生长在潮间带的重要海藻。虽然这种潮间带海藻已经成为了解胁迫耐受性和发育机制的实验系统，但适合qRT-PCR数据规范化的内参基因尚未被确定。在本研究中，6个候选内参基因，包括ACT、eIF4A、EF1α、GAPDH、TUA和UBQ（传统管家基因）的表达稳定性在代表不同发育阶段和胁迫条件的不同样本中得到验证。对geNorm、NormFinder和BestKeeper三种qRT-PCR分析方法进行系统评价。结果表明，在逆境条件下，ACT3、eIF4A和EF1α是叶藻的最佳内参基因；UBQ、EF1α和eIF4A适合用于研究紫杉树发育相关基因的表达。在胁迫条件和发育阶段，TUA的表达稳定性最低。本研究结果为利用qRT-PCR系统进行叶藻基因表达鉴定提供了参考基因应用指南。
Quantitative real-time PCR (qRT-PCR) is frequently used for gene expression analysis. The selection of reference genes is required for normalization of the variation to avoid misinterpretation of experimental results and erroneous analyses. Pyropia yezoensis, growing in the intertidal zone, is an economically important seaweed. Although this intertidal seaweed has been an experimental system for understanding stress tolerance and developmental mechanisms, reference genes suitable for the normalization of qRT-PCR data have not previously been identified. In this study, expression stability of six candidate reference genes, including ACT, eIF4A, EF1α, GAPDH, TUA, and UBQ (traditional housekeeping genes), has been validated in a diverse set of samples representing different developmental stages and stress conditions of P. yezoensis. Three qRT-PCR analysis methods, geNorm, NormFinder, and BestKeeper, were evaluated systematically. The results indicated that ACT3, eIF4A, and EF1α were the optimal reference genes for P. yezoensis under stress conditions; UBQ, EF1α, and eIF4A were suitable for studying the expression of genes related to P. yezoensis development. TUA showed the lowest expression stability both under stress conditions and over developmental stages. Our results have provided a reference gene application guideline for P. yezoensis gene expression characterization using the qRT-PCR system.