College of Horticulture, Sichuan Agricultural University, Chengdu, 611130, China.
Institute of Pomology and Olericulture, Sichuan Agricultural University, Chengdu, 611130, China.
BMC Mol Biol. 2018 Jun 22;19(1):8. doi: 10.1186/s12867-018-0109-4.
Strawberry has received much attention due to its nutritional value, unique flavor, and attractive appearance. The availability of the whole genome sequence and multiple transcriptome databases allows the great possibility to explore gene functions, comprehensively. Gene expression profiles of a target gene can provide clues towards the understanding of its biological function. Quantitative real-time PCR (qRT-PCR) is a preferred method for rapid quantification of gene expression. The accuracy of the results obtained by this method requires the reference genes with consistently stable expression to normalize its data.
In present study, the expression stability of seven candidate reference genes in diverse sample subsets of different tissues and fruit developmental stages, and plant subjected to light quality and low temperature treatments was evaluated using three statistical algorithms, geNorm, NormFinder, and BestKeeper. Our data indicated that the expression stability of reference genes varied under different experimental conditions. Overall, DBP, HISTH4, ACTIN1 and GAPDH expressed much more stably. PIRUV, ACTIN2 and 18S were not recommended for normalization in given experimental conditions due to low stability. In addition, the relative expression pattern of HY5 (ELONGATED HYPOCOTYL5) was conducted to further confirm the reliability of the reference genes, which demonstrated the correct adoption of reference genes was of great importance in qRT-PCR analysis.
Expression stability of reference genes from strawberry varied across selected experimental conditions. Systematic validation of reference genes prior to calculation of target gene expression level should be done to improve the accuracy and consistency of qRT-PCR analysis.
草莓因其营养价值、独特风味和诱人外观而备受关注。全基因组序列和多个转录组数据库的可用性使得全面探索基因功能成为可能。目标基因的基因表达谱可以为了解其生物学功能提供线索。定量实时 PCR（qRT-PCR）是快速定量基因表达的首选方法。该方法获得的结果的准确性需要具有一致稳定表达的参考基因来对其数据进行标准化。
在本研究中，使用三种统计算法 geNorm、NormFinder 和 BestKeeper，评估了七个候选参考基因在不同组织和果实发育阶段的不同样本子集以及植物受到光质和低温处理时的表达稳定性。我们的数据表明，参考基因的表达稳定性在不同的实验条件下存在差异。总体而言，DBP、HISTH4、ACTIN1 和 GAPDH 的表达更为稳定。由于稳定性低，PIRUV、ACTIN2 和 18S 不建议在给定的实验条件下用于归一化。此外，还进行了 HY5（伸长的下胚轴 5）的相对表达模式分析，以进一步证实参考基因的可靠性，这表明在 qRT-PCR 分析中正确采用参考基因对于提高准确性和一致性非常重要。
草莓参考基因的表达稳定性在所选实验条件下存在差异。在计算靶基因表达水平之前，应系统地验证参考基因，以提高 qRT-PCR 分析的准确性和一致性。
