VALIDATION OF REFERENCE GENES FOR RT-QPCR IN PORCINE OOCYTES CULTURED IN ANTIOXIDANT-ENRICHED MEDIA

Reference genes are essential for normalising data in RT-qPCR experiments and are critical in reducing technical variability. This study aimed to identify and validate stable reference genes for accurate gene expression analysis in porcine oocytes matured in culture media supplemented with antioxidants. Five commonly used reference genes were assessed: PPIA, RPL4, GAPDH, YWHAZ, and TBP, [2, 3] in oocytes cultured under oxidative stress conditions induced by Vitamin C. The experiment involved four experimental groups, analyzed in triplicate using SYBR Green RT-qPCR. PPIA exhibited the lowest SEM, indicating high precision and stability across different experimental conditions. GAPDH showed the most variability, confirming its unsuitability as a reference gene in this context. Differences between groups were statistically significant (p less tahn 0.01) for most genes, with some exceptions for PPIA, YWHAZ, and TBP in specific conditions. The gene stability was evaluated using the NormFinder algorithm, which provided a ranking based on the stability of gene expression across experimental groups. PPIA was found to be the most stable, demonstrating minimal variation in expression levels, while GAPDH was identified as the least stable gene. The findings emphasize the importance of selecting appropriate reference genes for each specific experimental condition to ensure the accuracy of gene expression data.