ANALYSIS OF HOUSEKEEPING GENE STABILIZATION IN ISOLATED MITOCHONDRIA

Öz

Objective: The mitochondria isolation method is used to investigate mitochondrial processes. RT-qPCR measurement of gene transcription levels is frequently used in these studies. In this method, it is important that the housekeeping genes used in the normalisation of the results are stable. The study aimed to determine which of two housekeeping genes is more stable in isolated mitochondria. Material and Method: Mitochondria were isolated from FHC cells. Membrane integrity and functionality were measured by mitotracker and JC-1 stainings and ROS ratio by flow cytometry. Housekeeping genes GAPDH and β-actin were used in RT-qPCR, and gene stability was calculated using ΔCq method and percentage coefficient of variance. For gene validation, CAT and SOD1 transcriptions were calculated by the 2-ΔΔCq method. These genes were selected because oxidative stress status has been detected. Result and Discussion: Isolated mitochondria membrane integrity was preserved and the membrane potential ratio was found 91%. ROS ratio was 1.6% and there was no oxidative stress that would affect the antioxidant enzymes levels. ΔCq values were 4.54±0.06 for β-actin and 0.69±0.16 for GAPDH, with 1.33% and 23.95% percentage coefficient of variance respectively. CAT transcription levels were 25.7±2.6 and 1.8±0.33, while SOD1 levels were 70.1±12.7 and 4.8±0.6 correspondingly. It was concluded that β-actin may be more stable than GAPDH. The potential impact of housekeeping gene selection on outcomes should be considered.

Anahtar Kelimeler

• Housekeeping gene stabilization
• isolated mitochondria
• RT-qPCR

İZOLE MİTOKONDRİLERDE HOUSEKEEPING GEN STABİLİZASYONUNUN ANALİZİ

Öz

Amaç: Mitokondri izolasyonu metodu mitokondriyal süreçlerin araştırılmasında kullanılır. Bu araştırmalarda gen transkripsiyon seviyelerinin RT-qPCR ölçümü sıklıkla kullanılmaktadır. Bu yöntemde sonuçların normalizasyonunda kullanılan housekeeping genlerin stabil olması önemlidir. Bu çalışma, izole mitokondride iki housekeeping genden hangisinin daha stabil olduğunu belirlemeyi amaçlamıştır. Gereç ve Yöntem: Mitokondriler FHC hücrelerinden izole edilmiştir. İzole mitokondrilerin membran bütünlüğü ve fonksiyonelliği mitotracker ve JC-1 boyamaları ile ROS oranı flow sitometriyle ölçülmüştür. RT-qPCR analizinde housekeeping gen olarak GAPDH ve β-aktin kullanılmış, gen stabilitesi ise ΔCq metodu ve yüzde varyans katsayısına göre hesaplanmıştır. Gen validasyonu için CAT ve SOD1 transkripsiyonları 2-ΔΔCq yöntemiyle hesaplanmıştır. Bu genler oksidatif stres durumunun tespit edilmiş olması nedeniyle seçilmiştir. Sonuç ve Tartışma: İzole mitokondrilerin membran bütünlüğünü koruduğu görülmüş, membran potansiyeli oranı ise %91 olarak bulunmuştur. ROS oranı %1.6 olarak ölçülmüştür ve buna göre antioksidan enzim düzeyini etkileyecek bir oksidatif stres olmadığı değerlendirilmiştir. ΔCq değerleri β-aktin için 4.54±0.06 ve GAPDH için 0.69±0.16 iken, yüzde varyans katsayısı sırasıyla %1,33 ve %23.95 idi. CAT transkripsiyon seviyesi, β-actin'e göre 25.7±2.6 ve GAPDH'a göre 1.8±0.33 iken, SOD1 seviyeleri β-aktin’e göre 70.1±12.7 ve GAPDH'a göre 4.8±0.6 olarak hesaplanmıştır. Sonuçlara göre β-aktin’in GAPDH’a göre daha stabil olabileceği kanısına varılmıştır. Analizlerde housekeeping gen seçiminin sonuçlar üzerindeki potansiyel etkisi dikkate alınmalıdır.

Anahtar Kelimeler

• Housekeeping gen stabilitesi
• izole mitokondri
• RT-qPCR

Kaynakça

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