Departement of Veterinary Clinical and Diagnostic Sciences, Faculty of Veterinary Medicine, University of Calgary, 3330 Hospital Drive NW, Calgary, Alberta T2N 4N1, Canada.
BMC Mol Biol. 2011 Jan 28;12:5. doi: 10.1186/1471-2199-12-5.
The stability of reference genes has a tremendous effect on the results of relative quantification of genes expression by quantitative polymerase chain reaction. Equine Inflammatory Airway Disease (IAD) is a common condition often treated with corticosteroids. The diagnosis of IAD is based on clinical signs and bronchoalveolar lavage (BAL) fluid cytology. The aim of this study was to identify reference genes with the most stable mRNA expression in the BAL cells of horses with IAD irrespective of corticosteroids treatment.
The expression stability of seven candidate reference genes (B2M, HPRT, GAPDH, ACTB, UBB, RPL32, SDHA) was determined by qRT-PCR in BAL samples taken pre- and post- treatment with dexamethasone and fluticasone propionate for two weeks in 7 horses with IAD. Primers' efficiencies were calculated using LinRegPCR. NormFinder, GeNorm and qBasePlus softwares were used to rank the genes according to their stability. GeNorm was also used to determine both the ideal number and the best combination of reference genes. GAPDH was found to be the most stably expressed gene with the three softwares. GeNorm ranked B2M as the least stable gene. Based on the pair-wise variation cut-off value determined with GeNorm, the number of genes required for optimal normalization was four and included GAPDH, SDHA, HPRT and RPL32.
The geometric mean of GAPDH, HPRT, SDHA and RPL32 is recommended for accurate normalization of quantitative PCR data in BAL cells of horses with IAD treated with corticosteroids. If only one reference gene can be used, then GAPDH is recommended.
参考基因的稳定性对通过定量聚合酶链反应相对定量基因表达的结果有巨大影响。马的炎症性气道疾病（IAD）是一种常见疾病，常使用皮质类固醇治疗。IAD 的诊断基于临床症状和支气管肺泡灌洗（BAL）液细胞学。本研究的目的是鉴定在 IAD 马的 BAL 细胞中，无论是否接受皮质类固醇治疗，具有最稳定 mRNA 表达的参考基因。
通过 qRT-PCR 测定了 7 匹马在接受两周地塞米松和丙酸氟替卡松治疗前后的 BAL 样本中 7 个候选参考基因（B2M、HPRT、GAPDH、ACTB、UBB、RPL32、SDHA）的表达稳定性。使用 LinRegPCR 计算引物效率。使用 NormFinder、GeNorm 和 qBasePlus 软件根据稳定性对基因进行排名。GeNorm 还用于确定理想的参考基因数量和最佳组合。根据三个软件，GAPDH 被认为是表达最稳定的基因。GeNorm 将 B2M 列为最不稳定的基因。根据 GeNorm 确定的成对变异截止值，最佳归一化所需的基因数为 4 个，包括 GAPDH、SDHA、HPRT 和 RPL32。
对于接受皮质类固醇治疗的 IAD 马的 BAL 细胞中的定量 PCR 数据，建议使用 GAPDH、HPRT、SDHA 和 RPL32 的几何平均值进行准确归一化。如果只能使用一个参考基因，则推荐使用 GAPDH。
