Sığır ovaryumunda korpus luteum, korteks ve medullada qPCR için uygun housekeeping genlerin belirlenmesi

Öz

Stabil referans genlerle doğru normalizasyon, güvenilir kantitatif PCR (qPCR) analizleri için kritik öneme sahiptir. Referans gen stabilitesi doku, gelişim evresi ve fizyolojik koşullara göre farklılık gösterir ve yanlış seçimi sonuçları yanıltabilir. Sığır ovaryum dokularında yaygın kullanılan ACTB ve GAPDH genlerinin stabil olmadığının gösterilmesi, alternatif adaylara duyulan ihtiyacı ortaya koymaktadır. Bu çalışmada, dört referans genin (PPIA, HSP90AB1, RPL37A, RPS17) stabilitesi sığır ovaryumunun farklı bölgelerinde (korteks, medulla, korpus luteum) değerlendirilmiştir. Ovaryum dokuları, altı sağlıklı inekten post-mortem olarak toplanmış, RNA izolasyonu ve cDNA sentezi yapılmıştır. Stabilite, geNorm, NormFinder ve BestKeeper algoritmalarıyla hem dokuya özgü hem de tüm ovaryum genelinde incelenmiştir. Tüm primerler yüksek verimlilik (%90–110) ve R² >0.99 değerleri göstermiştir. Bulgular dokuya özgü farklılıklar ortaya koymuştur: Kortekste RPL37A veya RPS17 en stabil gen olarak belirlenmiştir; medullada RPL37A ve RPS17 algoritmalar arasında tutarlı bulunmuştur; korpus luteumda RPL37A ve PPIA stabil kalmıştır. Tüm dokular birlikte değerlendirildiğinde RPL37A + PPIA en güvenilir normalizasyonu sağlamış, RPL37A genel olarak en stabil gen olmuştur. Buna karşılık HSP90AB1 instabil bulunmuş ve uygun değildir. Bununla birlikte, küçük örneklem büyüklüğü bu çalışmanın bir sınırlılığıdır. Bu bulgular, ribozomal genlerin (RPL37A, RPS17) sığır ovaryum dokularında PPIA’ya göre daha yüksek stabilite gösterdiğini ortaya koyarak, gen ekspresyon çalışmalarına metodolojik rehberlik sunmakta ve özellikle fizyolojik koşullar altında yürütülen reprodüktif biyoloji araştırmalarına değerli katkılar sağlamaktadır.

Anahtar Kelimeler

• Sığır reprodüksiyonu
• Gen stabilitesi
• Normalizasyon
• Referans gen
• RT-qPCR validasyonu

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

Abstract

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.

Keywords

• Bovine reproduction
• Gene stability
• Normalization
• Reference gene
• RT-qPCR validation

Kaynakça

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