| Summary: | TNFα, and IFNγ) were selected for analysis, and two validated housekeeping genes (PGK1 and RPL4) with stable expression were used as reference genes. Sixteen blood samples were obtained from four animals with different health conditions and stored in RNAlater solution. These samples were used for RNA extraction followed by qRT-PCR analysis. Analysis of gene transcripts was performed by relative quantitation using the comparative Cq method with the integration of amplification efficiency and two reference genes. The expression levels of each gene in the samples from clinically healthy animals were normally distributed. Transcript outliers for IL-2Rα, IL-4, IL-12, TNFα, and IFNγ were noticed in four samples collected from two clinically unhealthy animals. This assay has the potential to identify immune system deviation from normal state, which is caused by health problems. Furthermore, knowing the immune status of captive cetaceans could help both trainers and veterinarians in implementing preventive approaches prior to disease onset. 147 Shapiro-Wilk test showed that NV data of each gene was normally distributed (P > 0.05). 213 enhanced specificity was used in this study to prevent the limitations of SYBR Green assays. 214 Amplification efficiency and reference gene selection are two important factors in gene transcript 215 study using qPCR. The traditional NV is calculated using the ΔCq method (CqT -CqR).
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