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Stress factors during poultry production can evoke changes in gene transcription and protein synthesis in the hen oviduct and could affect the internal and external egg quality. Studies of relative gene expression require the identification of the most stable reference genes for the quantitative polymerase chain reaction (qPCR) to investigate the reproductive tissues' response in laying hens kept in different production systems. The objective of this study was to determine the most stable reference genes of the magnum tissues of laying hens housed in two different production systems. Hy‐Line Brown hens were reared under the same sanitary conditions until 15 weeks of age. Later on, they were transferred into two different production systems, conventional cage (CC) and cage free (CF), up to 82 weeks of age. At 50 and 60 weeks, a total of six hens from each production system were euthanized, and magnum samples were collected. The qPCR was used to determine the RNA transcription level of five reference genes,  ACTB ,  18S ,  GAPDH ,  MSX2  and  HMBS . These genes were evaluated for transcript expression in magnum tissues by NormFinder, BestKeeper, geNorm and RefFinder software. The results indicated that the most stable gene in the CF housing system was  HMBS  in three of the algorithms and in the CC housing system was the  18S , and the best combination of reference genes was  HMBS/GAPDH  in CF and  18S/HMBS  in CC. In conclusion,  HMBS ,  18S  and  GAPDH  genes could be used together as reference genes for the normalization of the magnum tissues transcript expression of hens in CF and CC housing systems.

Identification of reliable reference genes for expression studies in the magnum of laying hens housed in cage and cage‐free systems