ERCAN KURAR, Mehmet Osman ATLI, Aydın GÜZELOĞLU, YUSUF ÖZŞENSOY, Ahmet SEMACAN

Comparison of five different RNA isolation methods from equine endometrium for gene Transcription analysis

Bu çalışmanın amacı; kısrak endometrium biyopsi örneklerinden beş farklı RNA izolasyon metodu kullanılarak elde edilen RNA’nın; kalite ve miktarı ile Reverz Transkripsiyon-Polimeraz Zincir Reaksiyonu (RT-PZR) analizlerinde kullanılabilirliğinin araştırılmasıdır. Bu çalışmada; ticari olarak mevcut fenol/kloroform esasına dayanan üç farklı izolasyon kit (TRIzol, TRItidy ve EZRNA) ile kolon esasına dayanan iki farklı kit (UltraCleanTM ve E.Z.N.A.®) kullanılmıştır. Tüm kitlerden elde edilen total RNA’ların kalitesinin, gözlemlenen 28S ve 18S rRNA bantlarına göre iyi olduğu tespit edilmiştir. En düşük total RNA miktarı EZ-RNA kitinden elde edilmiştir. RNA örnekleri agaroz jel elektroforezinde kontrol edildiği zaman, kolon esasına dayanan kitlerde yüksek oranda genomik DNA (gDNA) kontaminasyonunun varlığı gözlemlenmiştir. Ancak, tüm kitlerden elde edilen total RNA’lardaki gDNA’lar DNase-I enzimi ile tamamen temizlenmiştir. Tüm kitlerden izole edilen RNA örneklerinden cDNA sentezlenmiş ve GAPDH geni primerleri kullanılarak PZR ile yükseltgenmiştir. Sonuçlar agaroz jel elektroforezinde kontrol edilmiştir. Sonuç olarak; tüm izolasyon kitlerinden iyi kalitede fakat farklı miktarlarda total RNA izole edilmiştir. RT-PZR gibi analizlerde kullanılmak istenen RNA örneklerinde gDNA kontaminasyonunun uzaklaştırılabilmesi için DNase-I enzimi ile muamele edilmesi önerilmektedir.

Anahtar Kelimeler:

biyopsi, RNA, tamamlayıcı DNA, endometriyum, gen ifadesi, genler, genom, at, izolasyon, kısrak, transkripsiyon

At endometriumunda gen transkripsiyon analiz çalışmalarında beş farklı RNA izolasyon yönteminin karşılaştırılması

In this study, five different isolation protocols to extract total RNA from biopsies of equine endometrium were compared in terms of quality and quantity of RNA samples with respect to downstream gene transcription analysis, such as Reverse Transcription-Polymerase Chain Reaction (RT-PCR). Three phenol-chloroform based protocols (TRIzol, TRItidy, EZ-RNA) and two column based protocols (UltraCleanTM and E.Z.N.A.®) that were commercially available were used. Each protocol yielded good quality total RNA and distinct 28S and 18S rRNA bands were observed in agarose gel electrophoreses. Amount of total RNA isolated was lower for EZ-RNA protocol. Column based protocols had RNA contaminated with great amount of genomic DNA, however, DNAse-I digestion was able to fully clean the DNA contamination from RNA in all the protocols used. Following cDNA synthesis and PCR, GAPDH, a housekeeping gene, bands were amplified from all the samples. In conclusion, all the protocols used extracted good quality but different amounts of total RNA and it is strongly recommended that RNA samples must undergo DNAse-I digestion before RT-PCR to eliminate gDNA contamination..

Keywords:

biopsy, RNA, complementary DNA, endometrium, gene expression, genes, genomes, horses, isolation, mares, transcription,

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