State Key Laboratory for Conservation and Utilization of Subtropical Agro-bioresources/Guangdong Key Laboratory for Postharvest Science and Technology, College of Horticulture, South China Agricultural University, Guangzhou, PR China.
PLoS One. 2012;7(8):e44405. doi: 10.1371/journal.pone.0044405. Epub 2012 Aug 31.
Real-time reverse transcription PCR (RT-qPCR) is a preferred method for rapid and accurate quantification of gene expression studies. Appropriate application of RT-qPCR requires accurate normalization though the use of reference genes. As no single reference gene is universally suitable for all experiments, thus reference gene(s) validation under different experimental conditions is crucial for RT-qPCR analysis. To date, only a few studies on reference genes have been done in other plants but none in papaya. In the present work, we selected 21 candidate reference genes, and evaluated their expression stability in 246 papaya fruit samples using three algorithms, geNorm, NormFinder and RefFinder. The samples consisted of 13 sets collected under different experimental conditions, including various tissues, different storage temperatures, different cultivars, developmental stages, postharvest ripening, modified atmosphere packaging, 1-methylcyclopropene (1-MCP) treatment, hot water treatment, biotic stress and hormone treatment. Our results demonstrated that expression stability varied greatly between reference genes and that different suitable reference gene(s) or combination of reference genes for normalization should be validated according to the experimental conditions. In general, the internal reference genes EIF (Eukaryotic initiation factor 4A), TBP1 (TATA binding protein 1) and TBP2 (TATA binding protein 2) genes had a good performance under most experimental conditions, whereas the most widely present used reference genes, ACTIN (Actin 2), 18S rRNA (18S ribosomal RNA) and GAPDH (Glyceraldehyde-3-phosphate dehydrogenase) were not suitable in many experimental conditions. In addition, two commonly used programs, geNorm and Normfinder, were proved sufficient for the validation. This work provides the first systematic analysis for the selection of superior reference genes for accurate transcript normalization in papaya under different experimental conditions.
实时逆转录聚合酶链式反应（RT-qPCR）是一种快速准确定量基因表达研究的首选方法。然而，RT-qPCR 的适当应用需要通过参考基因进行准确的归一化。由于没有单一的参考基因普遍适用于所有实验，因此在不同实验条件下验证参考基因对于 RT-qPCR 分析至关重要。迄今为止，只有少数关于其他植物的参考基因的研究，但在木瓜中没有。在本工作中，我们选择了 21 个候选参考基因，并使用三种算法（geNorm、NormFinder 和 RefFinder）在 246 个木瓜果实样本中评估了它们的表达稳定性。这些样本由 13 组不同实验条件下收集的样本组成，包括不同组织、不同储存温度、不同品种、发育阶段、采后成熟、气调包装、1-甲基环丙烯（1-MCP）处理、热水处理、生物胁迫和激素处理。结果表明，参考基因之间的表达稳定性差异很大，应根据实验条件验证不同的合适参考基因或参考基因组合用于归一化。一般来说，内参基因 EIF（真核起始因子 4A）、TBP1（TATA 结合蛋白 1）和 TBP2（TATA 结合蛋白 2）在大多数实验条件下表现良好，而最广泛使用的参考基因 ACTIN（肌动蛋白 2）、18S rRNA（18S 核糖体 RNA）和 GAPDH（甘油醛-3-磷酸脱氢酶）在许多实验条件下并不适用。此外，两种常用的程序 geNorm 和 Normfinder 被证明足以进行验证。这项工作为在不同实验条件下准确转录归一化选择优良的参考基因提供了第一个系统分析。
