Introduction: Real-time polymerase chain reaction (RT-qPCR) is an important tool for analyzing gene expression. However, before analyzing the expression of target genes, it is crucial to normalize the reference genes, in order to find the most stable gene to be used as an endogenous control. A gene that remains stable in all samples under different treatments is considered a suitable normalizer. In this sense, we aimed to identify stable reference genes for normalization of target genes in the heart and liver tissues from two genetically divergent groups of chickens (Cobb 500® commercial line and Peloco backyard chickens) under comfort and acute heat stress environmental conditions. Eight reference genes (ACTB, HPRT1, RPL5, EEF1, MRPS27, MRPS30, TFRC and LDHA) were analyzed for expression stability. The samples were obtained from 24 chickens, 12 from the backyard Peloco and 12 from the Cobb 500® line, exposed to two environmental conditions (comfort and heat stress). Comfort temperature was 23°C and heat stress temperature was 39.5°C for one hour. Subsequently, the animals were euthanized, and heart and liver tissue fragments were collected for RNA extraction and amplification. To determine the stability rate of gene expression, three different statistical algorithms were applied: BestKeeper, geNorm and NormFinder, and to obtain an aggregated stability list, the RankAgregg package of R software was used.
Results: The most stable genes using BestKeeper tool, including the two factors (genetic group and environmental condition), were LDHA, RPL5 and MRPS27 for heart tissue, and TFRC, RPL5 and EEF1 for liver tissue. Applying geNorm algorithm, the best reference genes were RPL5, EEF1 and MRPS30 for heart tissue and LDHA, EEF1 and RPL5 for liver. Using the NormFinder algorithm, the best normalizer genes were EEF1, RPL5 and LDHA in heart, and EEF1, RPL5 and ACTB in liver tissue. In the overall ranking obtained by RankAggreg package, considering the three algorithms, the RPL5, EEF1 and LDHA genes were the most stable for heart tissue, whereas RPL5, EEF1 and ACTB were the most stable for liver tissue.
Conclusion: According to the RankAggreg tool classification based on the three different algorithms (BestKeeper, geNorm and NormFinder), the most stable genes were RPL5, EEF1 and LDHA for heart tissue and RPL5, EEF1 and ACTB for liver tissue of chickens subjected to comfort and acute heat stress environmental conditions. However, the best reference genes may vary depending on the experimental conditions of each study, such as different breeds, environmental stressors, and tissues analyzed. Therefore, the need to perform priori studies to assay the best reference genes at the outset of each study is emphasized.