A Panel of Stably Expressed Reference Genes for Real-Time qPCR Gene Expression Studies of Mallards (<i>Anas platyrhynchos</i>)

Determining which reference genes have the highest stability, and are therefore appropriate for normalising data, is a crucial step in the design of real-time quantitative PCR (qPCR) gene expression studies. This is particularly warranted in non-model and ecologically important species for which appropriate reference genes are lacking, such as the mallard—a key reservoir of many diseases with relevance for human and livestock health. Previous studies assessing gene expression changes as a consequence of infection in mallards have nearly universally used β-actin and/or GAPDH as reference genes without confirming their suitability as normalisers. The use of reference genes at random, without regard for stability of expression across treatment groups, can result in erroneous interpretation of data. Here, eleven putative reference genes for use in gene expression studies of the mallard were evaluated, across six different tissues, using a low pathogenic avian influenza A virus infection model. Tissue type influenced the selection of reference genes, whereby different genes were stable in blood, spleen, lung, gastrointestinal tract and colon. β-actin and GAPDH generally displayed low stability and are therefore inappropriate reference genes in many cases. The use of different algorithms (GeNorm and NormFinder) affected stability rankings, but for both algorithms it was possible to find a combination of two stable reference genes with which to normalise qPCR data in mallards. These results highlight the importance of validating the choice of normalising reference genes before conducting gene expression studies in ducks. The fact that nearly all previous studies of the influence of pathogen infection on mallard gene expression have used a single, non-validated reference gene is problematic. The toolkit of putative reference genes provided here offers a solid foundation for future studies of gene expression in mallards and other waterfowl.筛选稳定性最优、适用于数据标准化的内参基因，是实时定量聚合酶链反应（real-time quantitative PCR，qPCR）基因表达研究设计中的关键步骤。对于缺乏合适内参基因的非模式生物及生态重要物种而言，这一步尤为必要，例如绿头鸭（mallard）——它是多种可影响人类与畜禽健康的疾病的关键宿主库。既往针对绿头鸭感染后基因表达变化的研究，几乎均以β-肌动蛋白（β-actin）和/或甘油醛-3-磷酸脱氢酶（GAPDH）作为内参基因，却未验证其作为标准化因子的适用性。若随意选择内参基因而未考量其在不同处理组间的表达稳定性，则可能导致数据解读出现偏差。本研究以低致病性甲型禽流感病毒感染模型为基础，针对绿头鸭基因表达研究中可用的11个候选内参基因，在6种不同组织中开展了稳定性评估。组织类型对内参基因的筛选存在显著影响：血液、脾脏、肺脏、胃肠道及结肠中稳定表达的内参基因各不相同。β-肌动蛋白（β-actin）与甘油醛-3-磷酸脱氢酶（GAPDH）整体稳定性较低，因此在多数场景下并不适合作为内参基因。不同算法（GeNorm与NormFinder）对内参基因的稳定性排名存在影响，但针对这两种算法，均可筛选出由两个稳定内参基因组成的组合，用于绿头鸭qPCR数据的标准化分析。本研究结果凸显了在开展鸭类基因表达研究前，验证内参基因选择合理性的重要性。既往所有探究病原体感染对绿头鸭基因表达影响的研究，均采用了未经过验证的单一内参基因，这一做法存在明显问题。本研究提供的候选内参基因工具包，可为未来绿头鸭及其他水禽的基因表达研究奠定坚实基础。