Sağlıklı FHC Hücrelerinde Mitokondri Transplantasyonunun Referans Gen Stabilizasyonu Üzerindeki Etkisi

Abstract

Amaç: Gen ekspresyon seviyelerinin belirlenmesinde RT-qPCR sıklıkla kullanılmaktadır. Ekspresyon seviyeleri tutarlı bir şekilde eksprese edilen referans genlere göre hesaplanır. Mitokondri transplantasyonu (MTx) çalışmalarında Gapdh ve Actb sık kullanılan genlerden olsa da gen seçimi konusunda kapsamlı araştırmaya rastlanılamamıştır. Gereç ve Yöntem: MTx ve mitokondri izolasyonu için sağlıklı Fetal İnsan Kolon (FHC) hücreleri kullanılmıştır. İzole mitokondrilerin protein, ATP, membran potansiyeli ve ROS ölçümleri ile kalite kontrolü yapılmıştır. MTx sonrası gen stabilitesini değerlendirmek ve doğrulamak için Gapdh, Actb, Sod ve Cat seviyeleri ölçülmüştür. Stabilite analizlerinde CV ve ΔCq yöntemlerinin yanında BestKeeper aracı kullanılmıştır. Bulgular: İzole mitokondri protein, ATP, MMP ve ROS düzeyleri sırasıyla 52.23 μg/mL, 533.58 nmol, %92 ve ROS %2 olarak ölçülmüştür. CV hesaplamasında Gapdh’nin Actb’den daha tutarlı olduğu görülmüştür (%2.15’e karşı %2.72). ΔCq yöntemi, Actb’nin (0.45’e karşı 0.46) biraz daha stabil olduğunu göstermiştir. Bestkeeper, Gapdh için varyasyonun daha az olduğunu (1.86%’ya karşı 2.20%), ancak Actb’nin Bestkeeper indeksine daha uyumlu (0.982’ye karşı 0.971) olduğunu göstermiştir. Farklı genlere Sod ve Cat ekspresyon seviyelerinin hesaplanmasının istatistiksel olarak anlamlı değişimlere yol açmıştır. Sonuç: Sonuçlara göre her iki gen de stabildir. Ancak Gapdh daha az değişkenlik gösterirken, Actb BestKeeper indeksi ile daha koreledir. GAPDH, düşük varyasyonu nedeniyle uygun bir seçimdir, ancak genlerin kombinasyonu daha güvenilir normalizasyon sağlayabilir. Araştırma bulguları referans gen seçiminin sonuçları etkileyebileceğini ve güvenilir sonuçlar alınması için referans gen stabilitesinin kontrol edilmelisi gerektiğini göstermektedir.

Keywords

• mitokondri transplantasyonu
• referans gen
• gen stabilitesi

Effect of Mitochondrial Transplantation on Reference Gene Stabilization in Healthy FHC Cells

Abstract

Objectives: RT-qPCR is frequently used to determine gene expression levels. Expression levels are calculated relative to consistently expressed reference genes. Although Gapdh and Actb are commonly used reference genes in mitochondrial transplantation (MTx) studies, no comprehensive research on gene selection has been found. Materials and Methods: Healthy Fetal Human Colon (FHC) cells were used for MTx and mitochondrial isolation. Quality control was performed by measuring protein, ATP, membrane potential, and ROS levels in isolated mitochondria. Gapdh, Actb, Sod, and Cat levels were measured after MTx to evaluate and validate gene stability. The CV and ΔCq methods were used in stability analyses, along with the BestKeeper tool. Results: Isolated mitochondrial protein, ATP, MMP, and ROS levels were measured as 52.23 μg/mL, 533.58 nmol, 92%, and ROS 2%, respectively. In CV calculations, Gapdh was found to be more consistent than Actb (2.15% versus 2.72%). The ΔCq method showed Actb to be slightly more stable (0.45 versus 0.46). The Bestkeeper found that the variation was lower for Gapdh (1.86% vs. 2.20%), but Actb was more consistent with the Bestkeeper index (0.982 vs. 0.971). Calculating the expression levels of Sod and Cat for different genes led to statistically significant changes. Conclusion: According to the results, both genes are stable. However, Gapdh shows less variability, while Actb is more correlated with the BestKeeper index. GAPDH is a proper choice due to its low variation, but a combination of genes may provide more reliable normalization. The research findings indicate that the choice of reference gene can affect the results and that the stability of the reference gene must be checked to obtain reliable results.

Keywords

• mitochondrial transplantation
• reference gene
• gene stabilization

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