The accuracy of quantitative real time PCR (RTqPCR) can be attained only when a suitable reference gene is used. The gene expression for a particular gene may vary within different cells at different conditions. Hence, the suitability and stability of various potential reference genes have to be determined for expression studies. In this study, we have examined the potential of four different reference genes including β-Actin (ACTB), 18S ribosomal RNA (18S), glyceraldehyde-3P-dehydrogenase (GAPDH), and elongation factor 1 alpha (EF1A_A) in seven different tissues including gill, liver, kidney, spleen, heart, muscle and intestine of goldfish (Carassius auratus). The housekeeping genes were analyzed from healthy fish and in CyHV-2 challenged fish. Based upon the real time PCR results the gene expression varied among the genes and in tissues. The expression levels of the housekeeping genes were then compared and evaluated with the RefFinder web tool which analyses results using four different algorithms – BestKeeper, delta Ct, geNorm and NormFinder. EF1A_A was ranked to be the best gene in healthy fish by BestKeeper and geNorm analysis. The delta Ct and NormFinder algorithm have found 18S to be a stable gene in healthy fish but 18S was given to be least expressed in challenged fish. ACTB was also given as a stable gene by geNorm analysis in both healthy and challenged fish. Also, in CyHV-2 challenged fish, EF1A_A was identified as the best gene by all the three analysis except by BestKeeper analysis, where it has ranked GADPH as the best housekeeping gene. Expression of the four candidate reference genes differed across all tissue types tested, inferring that a thorough study of the reference genes is necessary for cross tissue comparison. These results can be further used in the immune gene response study of goldfish infected with any viral pathogen to develop better health strategies in the disease management of goldfish aquaculture.

只有使用合适的参考基因，才能获得实时定量 PCR (RTqPCR) 的准确性。特定基因的基因表达在不同条件下的不同细胞内可能不同。因此，必须确定各种潜在参考基因的适用性和稳定性以进行表达研究。在这项研究中，我们检查了四种不同参考基因的潜力，包括 β-肌动蛋白 (ACTB)、18S 核糖体 RNA (18S)、甘油醛-3P-脱氢酶 (GAPDH) 和延伸因子 1 α (EF1A _A ) 在七种不同的金鱼 ( Carassius auratus) 的鳃、肝、肾、脾、心脏、肌肉和肠等组织）。对健康鱼和 CyHV-2 攻击鱼的管家基因进行了分析。基于实时PCR结果，基因表达在基因和组织中不同。然后使用 RefFinder 网络工具比较和评估看家基因的表达水平，该工具使用四种不同的算法分析结果——BestKeeper、delta Ct、geNorm 和 NormFinder。EF1A _A被 BestKeeper 和 geNorm 分析评为健康鱼中最好的基因。delta Ct 和 NormFinder 算法发现 18S 在健康鱼中是一个稳定的基因，但 18S 在受攻击的鱼中表达最少。ACTB 还通过 geNorm 分析在健康和受攻击的鱼中作为稳定基因给出。此外，在 CyHV-2 挑战的鱼中，EF1A _A除了通过 BestKeeper 分析将 GADPH 列为最佳看家基因外，所有三个分析都将其鉴定为最佳基因。四种候选参考基因的表达在所有测试的组织类型中都不同，因此需要对参考基因进行彻底研究以进行跨组织比较。这些结果可进一步用于感染任何病毒病原体的金鱼的免疫基因反应研究，以制定更好的金鱼养殖疾病管理健康策略。