Guangdong Key Laboratory for Postharvest Science, College of Horticultural Science, South China Agricultural University, Guangzhou, 510642, People's Republic of China.
Planta. 2011 Aug;234(2):377-90. doi: 10.1007/s00425-011-1410-3. Epub 2011 Apr 20.
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. The RPS2 and UBQ2 genes were validated as the most suitable reference genes across all tested samples. More importantly, our data further showed that the widely used reference genes, ACT and GAPDH, were not the most suitable reference genes in many banana sample sets. In addition, the expression of MaEBF1, 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.
逆转录定量实时 PCR（RT-qPCR）是一种灵敏的基因表达定量技术，但它的成功取决于用于数据归一化的参考基因（s）的稳定性。在果树中已经进行了一些关于参考基因验证的研究，但在香蕉中还没有。在本工作中，选择了 20 个候选参考基因，并使用 geNorm 和 NormFinder 两种算法评估和分析了这 144 个香蕉样本中的表达稳定性。这些样本由八个在不同实验条件下收集的样本集组成，包括不同的组织、发育阶段、采后成熟、胁迫（冷害、高温和病原体）和激素处理。结果表明，根据实验条件，应该选择不同的合适的参考基因（s）或参考基因组合进行归一化。RPS2 和 UBQ2 基因被验证为所有测试样本中最适合的参考基因。更重要的是，我们的数据进一步表明，在许多香蕉样本集中，广泛使用的参考基因 ACT 和 GAPDH 不是最适合的参考基因。此外，还使用了不同实验条件下感兴趣基因 MaEBF1 的表达来进一步确认验证的参考基因。总之，我们的结果为不同实验条件下参考基因（s）的选择提供了指导，并为更准确和广泛地在香蕉中使用 RT-qPCR 奠定了基础。
