Validation of Reference Genes for Real Time PCR Normalization in Milk Somatic Cells of Holstein Dairy Cattle

Real time-qPCR is the most reliable method for evaluation of mRNA expression levels. However, to obtain accurate results, selection of suitable reference genes is necessary for normalizing the real-time qPCR data. The aim of this research was to validate the expression stability of three potential reference genes (ACTB, GAPDH and UXT) in milk somatic cells of Holstein dairy cattle under different lactation stages. For this purpose two types of milk samples from eighteen healthy cows at three lactation stages (early, middle and late of lactation cycle) and four mastitic cows were included in this experiment. Total RNA was extracted from the milk somatic cells and then cDNA was synthesized. Real-time polymerase chain reaction (PCR) performed for ACTB, GAPDH and UXT genes as candidate reference genes. Then, the real-time PCR results were analyzed with BestKeeper program. The evaluation of selected genes by real-time PCR revealed that all genes were expressed in the healthy and mastitic dairy cows. In addition, the UXT and GADPH genes displayed the lowest and highest values of expression level, respectively. The ACTB gene was considered as the most suitable internal controls as it was stably expressed in milk somatic cells regardless of dairy cows conditions. Taken together, our results could help to select suitable reference gene for the normalization of expression levels in milk somatic cells of dairy cattle.