Bilge ÖZSAİT SELÇUK, Neslihan ÇOBAN, Dilek SEVER-KAYA, Sibel BULGURCUOĞLU-KURAN, Selva TÜRKÖLMEZ, Özlem DURAL

SPERMATOZOA VE EMBRİYOLARIN GEN EKSPRESYON ANALİZİ ÖNCESİNDE RNA İZOLASYONU VE HÜCRESEL RNA MİKTARLARININ BELİRLENMESİ

DOI: 10.26650/IUITFD.416666Amaç: Üreme biyolojisi araştırmalarında kullanılan örnekler genellikle, zor ulaşılabilir, nadir ve tekrarı olmayan hücre gruplarıdır. Diğer yandan, az sayıda hücre ile çalışıldığından dolayı, genetik analizlerde standart olarak kullanılan yöntemlerde modifikasyonlara ihtiyaç duyulmaktadır. Bu çalışmada amacımız, gen ekspresyonu araştırmalarında kullanılabilecek optimal düzeydeki total RNA’nın elde edilebilmesi amacı ile spermatozoa ve embriyo örneklerinden RNA izolasyon yönteminin geliştirilmesidir. Gereç ve Yöntem: Yardımla üreme tedavisi kapsamında kullanılmak amacı ile hazırlanan ve fertilizasyon işlemi gerçekleştirildikten sonra geriye kalan spermatozoa örneği diğer kontaminant hücrelerden saflaştırılarak dondurulmuştur. Diğer yandan, embriyo transferinden sonra geriye kalan preimplantasyon dönem embriyolar fosfat tamponlu tuz – polivinil alkol (PBS-PVA) karışımı içerisinde dondurularak saklanmıştır. Spermatozoa ve embriyolardan optimal düzeyde total RNA elde edilmesi amacı ile çeşitli optimizasyonlar yapılmıştır. Elde edilen total RNA’lar kaliteleri ve miktarları açısından değerlendirilmiştir. Spermatozoonda 28S ribozomal RNA (rRNA)’nın seçimli olarak degrade olmasından dolayı total RNA kalitesi, Bioanalyzer cihazı ile gerçekleştirilen analize ek olarak gerçek zamanlı kantitatif PCR ile gen amplifikasyonu yapılarak da kontrol edilmiştir.Bulgular: İzolasyonda yapılan uyarlamalardan sonra, takip eden gen ekspresyonu çalışmalarında kullanılabilecek kalitede ve miktarda total RNA elde edilmiştir. Ek olarak, örneklerden elde edilen hücresel total RNA miktarlarının önceki çalışmalar ile uyumlu olduğu belirlenmiştir. Sonuç: Daha sonra yapılması planlanan transkript profilleme araştırmaları için ön bir çalışma olarak gerçekleştirilen bu deneylerin sonucunda elde edilen bulgular, RNA izolasyon protokollerinde yapılan uyarlamaların etkin olduğunu göstermektedir.

Anahtar Kelimeler:

spermatozoon, preimplantasyon dönem embriyo

RNA ISOLATION AND DETECTION OF CELLULAR RNA QUANTITY OF SPERMATOZOA AND EMBRYOS PRIOR TO GENE EXPRESSION ANALYSES

DOI: 10.26650/IUITFD.416666Objective: Biological samples that are analyzed in reproductive biology are generally rare, difficult to obtain, and a nonreplicable group of cells. Furthermore, investigating low numbers of cells requires modifications to the routine methods used in genetic analyses. The aim of our study was to improve RNA isolation methods for obtaining a sufficient amount of total RNA from spermatozoa and embryo samples for downstream gene expression analyses.Materials and Methods: Excess spermatozoa samples (that had been prepared in the scope of assisted reproduction treatment) were purified of any contaminant cells and then frozen. Preimplantation stage embryos that had not been selected for embryo transfer were frozen in a phosphate-buffered saline–polyvinyl alcohol (PBS–PVA) solution. Modifications were done to obtain the optimal amount of total RNA from the spermatozoa and embryos, and subsequently both the quality and the quantity of the total RNA samples were assessed. Because of the selective degradation of 28S RNA samples in the spermatozoa, the total RNA quality was evaluated by gene amplification using quantitative real-time polymerase chain reaction (qRT-PCR) in addition to Bioanalyzer analysis.Results: Following the modifications to the isolation technique, a sufficient quality and quantity of total RNA were obtained from the spermatozoa and embryo samples, which could be used in downstream gene expression analyses. Furthermore, the amount of cellular total RNAs was consistent to that reported in previous studies. Conclusion: Results obtained from these experiments - conducted as a preliminary work for further transcript profiling studies - indicate that the modifications used in the RNA isolation techniques were effective.

Keywords:

Spermatozoon, preimplantation stage embryo,

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