Identification of suitable housekeeping genes for qPCR in the corpus luteum, cortex, and medulla of the bovine ovary

Accurate normalization with stable reference genes is critical for reliable quantitative PCR (qPCR). Reference gene stability differs by tissue, stage, and condition and inappropriate selection may bias results. In bovine ovarian tissues, commonly used genes such as ACTB and GAPDH have shown instability, emphasizing the need for alternative candidates. This study evaluated the stability of four reference genes (PPIA, HSP90AB1, RPL37A, RPS17) across bovine ovary regions (cortex, medulla, corpus luteum). Ovarian tissues were collected post-mortem from six healthy cows, and RNA isolation and cDNA synthesis were performed. Stability was assessed with geNorm, NormFinder, and BestKeeper, both tissue-specific and across the whole ovary. All primers showed high efficiency (90–110%) and R² >0.99. Results revealed tissue-specific variation: in the cortex, RPL37A or RPS17 was most stable; in the medulla, RPL37A and RPS17 were consistent across algorithms; in the corpus luteum, RPL37A and PPIA were stable. Across all tissues, RPL37A + PPIA provided the most reliable normalization, with RPL37A overall the most stable. In contrast, HSP90AB1 was unstable and unsuitable. However, the small sample size is a limitation of this study. These findings indicate that ribosomal genes (RPL37A, RPS17) are more stable than PPIA in bovine ovarian tissues, providing methodological guidance for accurate gene expression studies and contributing valuable insights to reproductive biology research, particularly under physiological conditions.