Zhang Shutao, Chen Chun, Xie Tingna, Ye Sudan
China Jiliang University, Zhejiang Provincial Key Laboratory of Biometrology and Inspection & Quarantine, Hangzhou, China.
Zhejiang Economic & Trade Polytechnic, Hangzhou, China.
PLoS One. 2017 Jul 3;12(7):e0179930. doi: 10.1371/journal.pone.0179930. eCollection 2017.
The selection of stable reference genes is a critical step for the accurate quantification of gene expression. To identify and validate the reference genes in Pandora neoaphidis-an obligate aphid pathogenic fungus-the expression of 13classical candidate reference genes were evaluated by quantitative real-time reverse transcriptase polymerase chain reaction(qPCR) at four developmental stages (conidia, conidia with germ tubes, short hyphae and elongated hyphae). Four statistical algorithms, including geNorm, NormFinder, BestKeeper and Delta Ct method were used to rank putative reference genes according to their expression stability and indicate the best reference gene or combination of reference genes for accurate normalization. The analysis of comprehensive ranking revealed that ACT1and 18Swas the most stably expressed genes throughout the developmental stages. To further validate the suitability of the reference genes identified in this study, the expression of cell division control protein 25 (CDC25) and Chitinase 1(CHI1) genes were used to further confirm the validated candidate reference genes. Our study presented the first systematic study of reference gene(s) selection for P. neoaphidis study and provided guidelines to obtain more accurate qPCR results for future developmental efforts.
选择稳定的内参基因是准确定量基因表达的关键步骤。为了鉴定和验证专性蚜虫病原真菌新蚜虫疠霉中的内参基因，通过定量实时逆转录聚合酶链反应（qPCR）在四个发育阶段（分生孢子、带芽管的分生孢子、短菌丝和伸长菌丝）评估了13个经典候选内参基因的表达。使用包括geNorm、NormFinder、BestKeeper和Delta Ct法在内的四种统计算法，根据推定内参基因的表达稳定性对其进行排名，并指出用于准确标准化的最佳内参基因或内参基因组合。综合排名分析表明，ACT1和18S在整个发育阶段是表达最稳定的基因。为了进一步验证本研究中鉴定的内参基因的适用性，使用细胞分裂控制蛋白25（CDC25）和几丁质酶1（CHI1）基因的表达来进一步确认经验证的候选内参基因。我们的研究首次对新蚜虫疠霉研究中的内参基因选择进行了系统研究，并为未来的发育研究获得更准确的qPCR结果提供了指导。
