Stability of Reference Genes in Rat Ovarian Tissue Under Different Cryopreservation Conditions: A Multialgorithm Assessment

Background: Accurate quantitative real-time PCR (qPCR) normalization for cryopreserved ovarian tissue requires stable reference genes; however, published reports on the stability of such reference genes remain contradictory. Methods: The mRNA expression levels of nine candidate reference genes were quantified using reverse-transcription quantitative PCR (RT-qPCR) in a rat ovarian tissue model subjected to four conditions (fresh control, slow freezing, vitrification, liquid nitrogen snap freezing). Stability was assessed using four algorithms (NormFinder, geNorm, BestKeeper, and the ΔCt method), with emphasis on intergroup variation to identify genes most suitable for cross-protocol comparison. Results: Cryopreservation significantly affected the expression of all candidate genes (ANOVA, p < 0.05). Notably, expression levels were downregulated in the vitrification group (group effect: from −1.043 to −3.089) and upregulated in the slow freezing group (group effect: from + 0.939 to + 1.577). The composite RefFinder ranking identified Rpl9 and Tbp as the most stable genes overall, whereas NormFinder identified Ppia (stability value S = 1.133) and Ywhaz ( S = 1.176) as the optimal pair for cross-protocol comparison. Specifically, Ppia exhibited the smallest slow freezing group effect (+0.939), and Ywhaz showed the smallest liquid nitrogen snap freezing group effect (+0.283). geNorm analysis confirmed that two reference genes are sufficient for reliable normalization (pairwise variation V2/3 = 0.108). Hprt1 and Gapdh were consistently ranked as the least stable genes. Conclusion: Ppia was most stable under slow freezing, while Ywhaz was most stable under liquid nitrogen snap freezing. Among the four conditions, the expression of the candidate genes was most stable under slow freezing, whereas the greatest transcriptional stress was observed under vitrification, making it a valuable model for cryopreservation damage assessment but less ideal for maintaining reference gene stability. This work provides a validated, purpose-built normalization tool for the rat ovarian cryopreservation model, enabling objective molecular evaluation and facilitating the optimization of cryopreservation techniques.