| Summary: | 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 <i>(actb</i>, <i>ywhaz</i>, <i>b2m</i>, <i>sdha</i>, and <i>18s rRNA</i>) at different physiological stages (alert and emergency) in three different cattle breeds. In this study, five genes (<i>actb</i>, <i>ywhaz</i>, <i>b2m</i>, <i>sdha</i>, and <i>18s rRNA</i>) 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 <i>actb</i>, <i>18SrRNA</i>, and <i>b2m</i> 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, <i>actb</i>, <i>b2m</i>, and <i>ywhaz</i> were the most stable reference genes for the Romosinuano breed.
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