Validation of reference genes for RT-qPCR studies of gene expression in banana fruit under different experimental conditions
Validation of reference genes for RT-qPCR studies of gene expression in banana fruit under different experimental conditions
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不同实验条件下香蕉果实基因表达 RT-qPCR 研究的参考基因验证
DOI：
10.1007/s00425-011-1410-3
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发表时间：
2011-08-01
期刊：
影响因子：
4.3
通讯作者：
Chen, Jian-ye
中科院分区：
文献类型：
--
作者：
Chen, Lei;Zhong, Hai-ying;Chen, Jian-ye
逆转录定量实时PCR（RT-qPCR）是一种定量基因表达的敏感技术，但其成功取决于用于数据标准化的参考基因的稳定性。在果树上进行的参考基因验证研究很少，在香蕉上的研究还没有。在本研究中，选择了20个候选参考基因，并使用两种算法，geNorm和Normandum的表达稳定性进行了评估和分析，在144个香蕉样品。在不同的试验条件下采集了8个样品组，包括不同的组织、发育阶段、采后成熟、胁迫（低温、高温、病原菌）和激素处理。我们的结果表明，不同的合适的参考基因（S）或参考基因的组合，应根据实验条件进行选择。RPS 2和UBQ 2基因被验证为所有测试样品中最合适的参考基因。更重要的是，我们的数据进一步表明，广泛使用的参考基因，ACT和GAPDH，在许多香蕉样品组中不是最合适的参考基因。此外，在不同的实验条件下，表达MaEBF 1，一个感兴趣的基因，在调节果实成熟起着重要作用，被用来进一步确认验证的参考基因。综上所述，我们的研究结果为在不同实验条件下选择参考基因提供了指导，并为RT-qPCR在香蕉中更准确和广泛的应用奠定了基础。
Reverse transcription quantitative real-time PCR (RT-qPCR) is a sensitive technique for quantifying gene expression, but its success depends on the stability of the reference gene(s) used for data normalization. Only a few studies on validation of reference genes have been conducted in fruit trees and none in banana yet. In the present work, 20 candidate reference genes were selected, and their expression stability in 144 banana samples were evaluated and analyzed using two algorithms, geNorm and NormFinder. The samples consisted of eight sample sets collected under different experimental conditions, including various tissues, developmental stages, postharvest ripening, stresses (chilling, high temperature, and pathogen), and hormone treatments. Our results showed that different suitable reference gene(s) or combination of reference genes for normalization should be selected depending on the experimental conditions. TheRPS2andUBQ2genes were validated as the most suitable reference genes across all tested samples. More importantly, our data further showed that the widely used reference genes,ACTandGAPDH,were not the most suitable reference genes in many banana sample sets. In addition, the expression ofMaEBF1, a gene of interest that plays an important role in regulating fruit ripening, under different experimental conditions was used to further confirm the validated reference genes. Taken together, our results provide guidelines for reference gene(s) selection under different experimental conditions and a foundation for more accurate and widespread use of RT-qPCR in banana.