Seda SÜSGÜN, İlker KARACAN, Emrah YÜCESAN

Tek Basamaklı Ters Transkripsiyon Kantitatif PZR Yönteminin miRNA Ekspresyon Analizleri için Optimizasyonu

Amaç: Bu çalışmada mikroRNA (miRNA) hedeflerinin spesifik ola-rak belirlenmesi ve ekspresyon ölçümünün yapılmasına yönelik tek basamaklı ters transkripsiyon kantitatif polimeraz zincir reaksiyonu (RT-qPZR) yönteminin, seçilen iki farklı miRNA (hsa-miR-145-5p ve hsa-miR-146a-5p) için araştırılması ve sürecin optimizasyonu amaçlanmıştır.Gereç ve Yöntem: RNA eldesi HEK293T hücre hattından yapılmıştır. Çalışmada seçilen her iki miRNA hedefi için uygun primerler tasarlanmış, tek basamaklı RT-qPZR yöntemi ile optimizasyon işlemi gerçekleştirilmiştir. Hedef amplikonların spesifiklik doğrulamaları agaroz jel elektroforezi ve konvansiyonel dizileme yöntemi ile gerçekleştirilmiştir. Bulgular: Tek basamakta gerçekleştirilen RT-qPZR çalışmasının her iki primer için de yüksek spesifiklikte ve hassasiyette sonuç verdiği qPZR erime eğrisi analizi ve agaroz jel görüntüleme sistemi ile gös-terilmiştir. qPZR sırasındaki bağlanma sıcaklıklarından en düşük Ct değerinin 54°C’de elde edildiği görülmüştür. Ayrıca konvansiyonel Sanger dizileme sonucunda yalnızca ilgili miRNA dizilerinin hedef-lendiği ve spesifik olmayan herhangi bir çoğaltma işleminin olmadığı gösterilmiştir.Sonuç: Sunulan çalışmada deneysel tasarım her iki miRNA hedefi için de optimize edilerek spesifik olarak yalnızca hedef miRNA mo-lekülünün tespitinin yapılabildiği gösterilmiştir. Bu yaklaşım, ilerleyen çalışmalarda miRNA tespit ve ekspresyon analizlerinde güvenilir olarak kullanılabilecektir. Sunulan yaklaşım, düşük maliyetli, zamandan ve iş gücünden tasarruf sağlayan bir alternatif olması sebebiyle benzer tüm çalışmalarda değerlendirilebilir.

Anahtar Kelimeler:

miRNA, RT-qPZR, miR-145, miR-146a

Optimization of One Step Reverse Transcription Quantitative PCR Method for miRNA Expression Analyses

Objective: We aimed to investigate and optimize the one step re-verse transcription quantitative polymerase chain reaction (RT-qP-CR) method for specific detection and quantitation of two selected microRNA (miRNA)s, namely hsa-miR-145-5p and hsa-miR-146a-5p.Material and Method: RNA was extracted from HEK293T cell line. Primers were designed and experimentally optimized to be compatible with with one step RT-qPCR method for two selected miRNAs. Targeted amplicons were visualized with agarose gel electrophoresis and sequenced using the Sanger method for specificity verification.Results: High specificity of one step RT-qPCR amplification was demonstrated using melt curve and agarose gel electrophoresis analyses for both miRNA targets. It was shown that the earliest cycle threshold (Ct) values were obtained at the annealing tem-perature of 54°C. Also, target specificity was confirmed by con-ventional Sanger sequencing.Conclusion: In this study, one-step RT-qPCR design was optimized for both miRNA targets and target specificity was verified. Our study showed this approach to be a good candidate for miRNA detection and quantitation as a cost-effective alternative method. Furthermore, the approach is highly suitable for research projects as it is both low-cost and fast, involving less hands-on time.

Keywords:

miRNA, RT-qPCR, miR-145, miR-146a,

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