Ovine pulmonary adenocarcinoma (OPA) is a globally occurring tumor of lung epithelium which seriously affects the development of sheep farming. In our research, lung tissues of 3 naturally infected OPA individuals and 3 healthy individuals (2–4 years old) were collected. RNA was extracted for transcriptome analysis and reference gene selection. According to transcriptome analysis, 7 candidate reference genes (eukaryotic translation initiation factor 1, EIF1; glyceraldehyde-3-phosphate dehydrogenase, GAPDH; beta-actin, ACTB; GABA Type A receptor-associated protein, GABARAP; activating transcription factor 4, ATF4; ribosomal protein S15, RPS15; and Y-Box binding protein 1, YBX1) showed fragments per kilobase of transcript per million fragments mapped (FPKM) values > 200.0 and standard errors of the means (SEM) < 20.0. Expression of the above candidate reference genes was evaluated by Real-time quantitative polymerase chain reaction (RT-qPCR) combined with the analysis using GeNorm, NormFinder, and BestKeeper software. Comprehensive analysis of the results showed that ACTB was the most stable one, followed by EIF1 and GABARAP. Then, expression stability of the above three genes were validated, suggesting as suitable reference genes in sheep lung tissue, in additional 30 OPA-affected lung tissues and 10 healthy ovine lung tissues. Finally, our findings will be helpful for the subsequent study on the tumorigenic mechanism of OPA.

绵羊肺腺癌（OPA）是一种全球发生的肺上皮肿瘤，严重影响绵羊饲养业的发展。在我们的研究中，收集了3个自然感染的OPA个体和3个健康的个体（2-4岁）的肺组织。提取RNA用于转录组分析和参考基因选择。根据转录组分析，有7个候选参考基因（真核翻译起始因子1，EIF1； 3-磷酸甘油醛脱氢酶，GAPDH；β-肌动蛋白，ACTB； GABA A型受体相关蛋白，GABARAP；激活转录因子4，ATF4；GABA 。核糖体蛋白S15，RPS15和Y-Box结合蛋白1，YBX1）显示每千个碱基对的片段，每百万个片段的映射图谱（FPKM）值> 200.0，平均值的标准误（SEM）<20.0。通过实时定量聚合酶链反应（RT-qPCR），结合使用GeNorm，NormFinder和BestKeeper软件进行的分析，评估上述候选参考基因的表达。对结果的综合分析表明，ACTB是最稳定的，其次是EIF1和GABARAP。然后，验证了上述三个基因的表达稳定性，表明其在绵羊肺组织，另外30个受OPA影响的肺组织和10个健康的绵羊肺组织中是合适的参考基因。最后，我们的发现将有助于后续研究OPA的致瘤机制。