PPIA, HPRT1, and YWHAZ are suitable reference genes for quantitative polymerase chain reaction assay of the hypothalamic–pituitary–gonadal axis in sows

Abstract

Objective: The quantitative reverse transcription polymerase chain reaction (qPCR) is the most accurate and reliable technique for analysis of gene expression. Endogenous reference genes (RGs) have been used to normalize qPCR data, although their expression may vary in different tissues and experimental conditions. Verification of the stability of RGs in selected samples is a prerequisite for reliable results. Therefore, we attempted to identify the most stable RGs in the hypothalamic–pituitary–gonadal (HPG) axis in sows.Methods: The cycle threshold values of nine commonly used RGs (<i>18S</i>, <i>HPRT1</i>, <i>GAPDH</i>, <i>RPL4</i>, <i>PPIA</i>, <i>B2M</i>, <i>YWHAZ</i>, <i>ACTB</i>, and <i>SDHA</i>) from HPG axis-related tissues in the domestic sows in the different stages of estrus cycle were analyzed using two RG-finding programs, geNorm and Normfinder, to rank the stability of the pool of RGs. In addition, the effect of the most and least stable RGs was examined by normalization of the target gene, gonadotropin-releasing hormone (<i>GnRH</i>), in the hypothalamus.Results: <i>PPIA</i>, <i>HPRT1</i>, and <i>YWHAZ</i> were the most stable RGs in the HPG axis-related tissues in sows regardless of the stages of estrus cycle. In contrast, traditional RGs, including <i>18S</i> and <i>ACTB</i>, were found to be the least stable under these experimental conditions. In particular, in the normalization of <i>GnRH</i> expression in the hypothalamus against several stable RGs, <i>PPIA</i>, <i>HPRT1</i>, and <i>YWHAZ</i>, could generate significant (p<0.05) elevation of <i>GnRH</i> in the preovulatory phase compared to the luteal phase, but the traditional RGs with the least stability (<i>18S</i> and <i>ACTB</i>) did not show a significant difference between groups.Conclusion: These results indicate the importance of verifying RG stability prior to commencing research and may contribute to experimental design in the field of animal reproductive physiology as reference data.