Identification of Reference Genes for Expression Studies in the Whole-Blood from Three Cattle Breeds under Two States of Livestock Weather Safety

SIMPLE SUMMARY: Reductions in the fertility, body weight, and growth rate of cattle across the world are associated with the global warming phenomenon. Developing optimal management strategies is an important aspect of breeding programs for different breeds. Blood tissue undergoes dramatic physiological and metabolic changes during heat stress conditions, which involves the expression and regulation of a great number of genes across species. Real-time quantitative PCR (qPCR) is a method for the rapid and reliable quantification of mRNA transcription. Reference genes are used to normalize mRNA levels between different samples. Thus, the selection of high-quality reference genes is necessary for the interpretation of data generated by real-time PCR. ABSTRACT: Real-time PCR is widely used to study the relative abundance of mRNA due to its specificity, sensitivity, and repeatability quantification. However, relative quantification requires a reference gene, which should be stable in its expression, showing lower variation by experimental conditions or tissues. The aim of this study was to evaluate the stability of the expression of five commonly used reference genes (actb, ywhaz, b2m, sdha, and 18s rRNA) at different physiological stages (alert and emergency) in three different cattle breeds. In this study, five genes (actb, ywhaz, b2m, sdha, and 18s rRNA) were selected as candidate reference genes for expression studies in the whole blood from three cattle breeds (Romosinuano, Gyr, and Brahman) under heat stress conditions. The transcription stability of the candidate reference genes was evaluated using geNorm and NormFinder. The results showed that actb, 18SrRNA, and b2m expression were the most stable reference genes for whole blood of Gyr and Brahman breeds under two states of livestock weather safety (alert and emergency). Meanwhile, actb, b2m, and ywhaz were the most stable reference genes for the Romosinuano breed.