Biotic and abiotic stress affects the stability of housekeeping genes in Catla Labeo catla

The choice of a stable reference gene plays a crucial role in gene expression analysis. In most cases, the choice of housekeeping gene (HKG) is quite random. However, the expression of HKGs varies under different experimental conditions and in different tissues. The study focused on three reference genes in Catla Labeo catla—beta actin (β‐actin), glyceraldehyde‐3‐phosphate dehydrogenase (GAPDH), and elongation factor‐1 alpha (EF‐1α)—under four experimental conditions: bacterial challenge, hypoxia, a combination of bacterial challenge and hypoxia, and control. The fish were subjected to the above‐mentioned experimental circumstances, and five tissues (blood, gill, liver, kidney, and spleen) were collected at regular intervals. The investigation of gene expression in the five tissues was performed using real‐time (RT) quantitative polymerase chain reaction (qPCR), and the resultant cycle threshold values were subjected to several statistical techniques via the RefFinder tool to determine the most stable HKG. The results were ordered in accordance with the stability of the reference genes. In this study, GAPDHwas found to be the least stable gene, while β‐actinand EF‐1α remained stable in the kidney and liver under various circumstances. Other tissues demonstrated tissue‐ and treatment‐specific stability for the various HKGs. The findings indicate that GAPDHshould be avoided in Catla for gene expression studies and that different algorithms should be employed to validate the stability of HKGs in RT‐qPCR analysis. Impact statementThe current study provides insight on how to choose the housekeeping gene (HKG) appropriately in various experimental setups. The HKG expression is essential for optimizing real‐time quantitative polymerase chain reaction results. Nonetheless, the expression differs depending on the experimental setup and treatment groups. As a result, it is necessary to assess the stability of HKGs using various techniques. Our research will be useful in the selection of a stable HKG for gene expression investigations under hypoxic and bacterial challenge circumstances. The current study provides insight on how to choose the housekeeping gene (HKG) appropriately in various experimental setups. The HKG expression is essential for optimizing real‐time quantitative polymerase chain reaction results. Nonetheless, the expression differs depending on the experimental setup and treatment groups. As a result, it is necessary to assess the stability of HKGs using various techniques. Our research will be useful in the selection of a stable HKG for gene expression investigations under hypoxic and bacterial challenge circumstances.