Sağlıklı FHC Hücrelerinde Mitokondri Transplantasyonunun Referans Gen Stabilizasyonu Üzerindeki Etkisi

Year 2025, Volume: 78 Issue: 4, 358 - 366, 31.12.2025

Öner Ülger , Mualla Pınar Elçi , Tuğba Fatsa , Sema Ören , Meral Sarper

https://doi.org/10.65092/autfm.1782697

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

References

• Curis E, Nepost C, Grillault Laroche D, Courtin C, Laplanche J-L, Etain B, et al. Selecting reference genes in RT-qPCR based on equivalence tests: a network based approach. Sci Rep. 2019;9(1):16231.
• Joshi CJ, Ke W, Drangowska-Way A, O'Rourke EJ, Lewis NE. What are housekeeping genes? PLoS Comput Biol. 2022;18(7):e1010295.
• Chen C, Wu J, Hua Q, Tel-Zur N, Xie F, Zhang Z, et al. Identification of reliable reference genes for quantitative real-time PCR normalization in pitaya. Plant Methods. 2019;15(1):70.
• Davies KA, Welch SR, Sorvillo TE, Coleman-McCray JD, Martin ML, Brignone JM, et al. Optimal reference genes for RNA tissue analysis in small animal models of hemorrhagic fever viruses. Sci Rep. 2023;13(1):19384.
• van de Moosdijk AAA, van Amerongen R. Identification of reliable reference genes for qRT-PCR studies of the developing mouse mammary gland. Sci Rep. 2016;6(1):35595.
• Sumbria D, Berber E, Mathayan M, Rouse BT. Virus infections and host metabolism—can we manage the interactions? Front Immunol. 2021;11:594963.
• Silver N, Best S, Jiang J, Thein SL. Selection of housekeeping genes for gene expression studies in human reticulocytes using real-time PCR. BMC Mol Biol. 2006;7(1):33.
• Boda E, Pini A, Hoxha E, Parolisi R, Tempia F. Selection of Reference Genes for Quantitative Real-time RT-PCR Studies in Mouse Brain. J Mol Neurosci. 2009;37(3):238-53.
• Vandesompele J, De Preter K, Pattyn F, Poppe B, Van Roy N, De Paepe A, et al. Accurate normalization of real-time quantitative RT-PCR data by geometric averaging of multiple internal control genes. Genome Biol. 2002;3:1-12.
• Andersen CL, Jensen JL, Ørntoft TF. Normalization of Real-Time Quantitative Reverse Transcription-PCR Data: A Model-Based Variance Estimation Approach to Identify Genes Suited for Normalization, Applied to Bladder and Colon Cancer Data Sets. Cancer Res. 2004;64(15):5245-50.
• Pfaffl MW, Tichopad A, Prgomet C, Neuvians TP. Determination of stable housekeeping genes, differentially regulated target genes and sample integrity: BestKeeper--Excel-based tool using pair-wise correlations. Biotechnol Lett. 2004;26(6):509-15.
• Xie F, Xiao P, Chen D, Xu L, Zhang B. miRDeepFinder: a miRNA analysis tool for deep sequencing of plant small RNAs. Plant Mol Biol. 2012;80(1):75-84.
• Sundaram VK, Sampathkumar NK, Massaad C, Grenier J. Optimal use of statistical methods to validate reference gene stability in longitudinal studies. PLOS ONE. 2019;14(7):e0219440.
• Wang L, Zhou X, Lu T. Role of mitochondria in physiological activities, diseases, and therapy. Mol Biomed. 2025;6(1):42.
• Eş I, Ulger O. Organoids as 3D Models for Studying Exogenous Mitochondrial Transplantation. Cham: Springer International Publishing. p. 1-19.
• Ulger O, Eş I, Proctor CM, Algin O. Stroke studies in large animals: Prospects of mitochondrial transplantation and enhancing efficiency using hydrogels and nanoparticle-assisted delivery. Ageing Res Rev. 2024;100:102469.
• Lin R-Z, Im G-B, Luo AC, Zhu Y, Hong X, Neumeyer J, et al. Mitochondrial transfer mediates endothelial cell engraftment through mitophagy. Nature. 2024;629(8012):660-8.
• Wu Z, Chen L, Guo W, Wang J, Ni H, Liu J, et al. Oral mitochondrial transplantation using nanomotors to treat ischaemic heart disease. Nat Nanotechnol. 2024;19(9):1375-85.
• Zhang A, Liu Y, Pan J, Pontanari F, Chia-Hao Chang A, Wang H, et al. Zhang A, Liu Y, Pan J, Pontanari F, Chia-Hao Chang A, Wang H, et al. Delivery of mitochondria confers cardioprotection through mitochondria replenishment and metabolic compliance. Mol Ther. 2023;31(5):1468-79.
• Ülger Ö, Fatsa T, Oren S, Elçi MP. Analysis of housekeeping gene stabilization in isolated mitochondria. J Fac Pharm Ankara. 2025;49(2):320-8.
• Bian Z, Sun X, Liu L, Qin Y, Zhang Q, Liu H, et al. Sodium Butyrate Induces CRC Cell Ferroptosis via the CD44/SLC7A11 Pathway and Exhibits a Synergistic Therapeutic Effect with Erastin. Cancers (Basel). 2023;15(2).
• Zhou J, Yang Y, Wang Y-L, Zhao Y, Ye W-J, Deng S-Y, et al. Enhancer of zeste homolog 2 contributes to apoptosis by inactivating janus kinase 2/ signal transducer and activator of transcription signaling in inflammatory bowel disease. World J Gastroenterol. 2021;27(22):3073-84.
• Kim MJ, Hwang JW, Yun C-K, Lee Y, Choi Y-S. Delivery of exogenous mitochondria via centrifugation enhances cellular metabolic function. Sci Rep. 2018;8(1):3330.
• Lee JM, Hwang JW, Kim MJ, Jung SY, Kim K-S, Ahn EH, et al. Mitochondrial Transplantation Modulates Inflammation and Apoptosis, Alleviating Tendinopathy Both In Vivo and In Vitro. Antioxidants. 2021;10(5):696.
• Bodenstein DF, Powlowski P, Zachos KA, El Soufi El Sabbagh D, Jeong H, Attisano L, et al. Optimization of differential filtration-based mitochondrial isolation for mitochondrial transplant to cerebral organoids. Stem Cell Res Ther. 2023;14(1):202.
• Lee AR, Woo JS, Lee SY, Na HS, Cho KH, Lee YS, et al. Mitochondrial Transplantation Ameliorates the Development and Progression of Osteoarthritis. Immune Netw. 2022;22(2):e14.
• Chang ES, Song K, Song J-Y, Sung M, Lee M-S, Oh JH, et al. Real-time assessment of relative mitochondrial ATP synthesis response against inhibiting and stimulating substrates (MitoRAISE). Cancer Metab. 2024;12(1):25.
• Hwang JW, Lee MJ, Chung TN, Lee HAR, Lee JH, Choi SY, et al. The immune modulatory effects of mitochondrial transplantation on cecal slurry model in rat. Crit Care. 2021;25(1):20.
• Wang Z, Zhu J, Xu M, Ma X, Shen M, Yan J, et al. Transplantation of exogenous mitochondria mitigates myocardial dysfunction after cardiac arrest. eLife Sciences Publications, Ltd; 2025.
• Zhang Y, Wong HS. Are mitochondria the main contributor of reactive oxygen species in cells? J Exp Biol. 2021;224(5).
• McCully JD, Cowan DB, Pacak CA, Toumpoulis IK, Dayalan H, Levitsky S. Injection of isolated mitochondria during early reperfusion for cardioprotection. Am J Physiol Heart Circ Physiol. 2009;296(1):H94-H105.
• Grazioli S, Pugin J. Mitochondrial Damage-Associated Molecular Patterns: From Inflammatory Signaling to Human Diseases. Front Immunol. 2018;Volume 9 - 2018.
• Mehta R, Birerdinc A, Hossain N, Afendy A, Chandhoke V, Younossi Z, et al. Validation of endogenous reference genes for qRT-PCR analysis of human visceral adipose samples. BMC Mol Biol. 2010;11(1):39.
• Linardić M, Braybrook SA. Identification and selection of optimal reference genes for qPCR-based gene expression analysis in Fucus distichus under various abiotic stresses. PLoS One. 2021;16(4):e0233249.