Sıçanlarda Dokuya Özel Referans Genler: Kardiyak, Kas ve İskelet Sistemlerine Bakış

Abstract

mRNA ekspresyon çalışmalarında doğru normalizasyon, stabil referans genlerin seçilmesine bağlıdır. Bu derleme, normal ve çeşitli deneysel koşullar altında sıçan modellerinde kalp, kas ve kemik dokuları arasında referans gen önerilerini ortaya koymaktadır. 2000-2024 yılları arasında bildirilen çalışmalar, erkek sıçanların daha sık kullanıldığını ve Wistar ve Sprague-Dawley sıçanlarına odaklandığını göstermektedir. Çalışmalar sonucunda, sık kullanılan referans genlerin (GAPDH ve ACTB gibi) sınırlılıkları olduğu kanıtlanmıştır. geNorm, NormFinder ve BestKeeper gibi gelişmiş algoritmalar, stabilite kavramını belirlemekte ve kullanıcılar için serbestçe kullanılabilir durumdadır. Çalışmalar, referans gen seçiminde bölgesel ve zamansal faktörlerin önemini vurgulamaktadır. Ayrıca, cinsiyet ve gelişimsel aşama farklılıkları sıklıkla göz ardı edilmiştir ve bu anlamda literatürde hala bir boşluk vardır. Bu derleme, RT-PCR çalışmalarında güvenilirliği ve tekrarlanabilirliği sağlamada referans genlerin kritik rolünün altını çizmektedir. Farklı hastalık modellerinde normalizasyon için referans genlerin stabilitesine ve seçimine katkıda bulunacaktır. 12 öncü çalışmadan elde edilen bulguların sentezlenmesi, belirli dokular ve deneysel koşullar için sağlam referans genlerin seçilmesi için mevcut verileri sağlayarak, mRNA ekspresyon çalışmalarında gen ekspresyonunun doğru yorumlanmasına katkıda bulunacaktır.

Keywords

• Referans gen
• mRNA ekspresyonu
• RT-PCR
• Kalp
• Kas
• Kemik

Tissue-Specific Reference Genes in Rats: Insights into Cardiac, Muscular, and Skeletal Systems

Abstract

Correct normalization in mRNA expression studies depends on selecting stable reference genes. This review reveals reference gene suggestions among cardiac, muscle, and bone tissues in rat models under normal and various experimental conditions. Studies reported between 2000-2024 show that male rats were used more frequently and focused on Wistar and Sprague-Dawley rats. As a result of the studies, it has been proven that frequently used reference genes (such as GAPDH and ACTB) have limitations. Advanced algorithms such as geNorm, NormFinder, and BestKeeper determine the concept of stability and are freely available to users. Studies emphasize the importance of regional and temporal factors in reference gene selection. Moreover, gender and developmental stage differences have often been overlooked, and there is still a gap in the literature in this sense. This review underlines the critical role of reference genes in ensuring reliability and reproducibility in RT-PCR studies. It will contribute to the stability and selection of reference genes for normalization in different disease models. Synthesizing the findings from 12 pioneering studies will contribute to the accurate interpretation of gene expression in mRNA expression studies by providing existing data for selecting robust reference genes for specific tissues and experimental conditions.

Keywords

• reference gene
• mRNA expression
• RT-PCR
• Heart
• Muscle
• Bone

References

1. 1. Mony, T.J., M. Hong, and H.J. Lee, Empathy Study in Rodent Model of Autism Spectrum Disorders. Psychiatry Investig, 2018. 15(2): p. 104-110.
2. 2. Sivaselvachandran, S., et al., Behavioral and mechanistic insight into rodent empathy. Neurosci Biobehav Rev, 2018. 91: p. 130-137.
3. 3. Kolb, B. and G.C. Teskey, Age, experience, injury, and the changing brain. Dev Psychobiol, 2012. 54(3): p. 311-25.
4. 4. Ben-Ami Bartal, I., The complex affective and cognitive capacities of rats. Science, 2024. 385(6715): p. 1298-1305 . 5. Mukherjee, P., et al., Role of animal models in biomedical research: a review. Lab Anim Res, 2022. 38(1): p. 18.
5. 6. Eisenberg, E. and E.Y. Levanon, Human housekeeping genes, revisited. Trends Genet, 2013. 29(10): p. 569-74.
6. 7. Butte, A.J., V.J. Dzau, and S.B. Glueck, Further defining housekeeping, or "maintenance," genes Focus on "A compendium of gene expression in normal human tissues". Physiol Genomics, 2001. 7(2): p. 95-6.
7. 8. Zhang, W.X., et al., Selection of Suitable Reference Genes for Quantitative Real-Time PCR Normalization in Three Types of Rat Adipose Tissue. Int J Mol Sci, 2016. 17(6).
8. 9. Goncu, B., Identification of suitable reference genes for RT-qPCR studies in human parathyroid tissue glandular cells. Gene, 2024. 912: p. 148380.

9. 10. Svingen, T., et al., Selection of reference genes for quantitative RT-PCR (RT-qPCR) analysis of rat tissues under physiological and toxicological conditions. PeerJ, 2015. 3: p. e855.
10. 11. Lowe, D.A., et al., Glyceraldehyde-3-phosphate dehydrogenase varies with age in glycolytic muscles of rats. J Gerontol A Biol Sci Med Sci, 2000. 55(3): p. B160-4.
11. 12. Sun, J.H., et al., Validation of reference genes for estimating wound age in contused rat skeletal muscle by quantitative real-time PCR. Int J Legal Med, 2012. 126(1): p. 113-20. 13. Vesentini, N., et al., Selection of reference genes in different myocardial regions of an in vivo ischemia/reperfusion rat model for normalization of antioxidant gene expression. BMC Res Notes, 2012. 5: p. 124.
12. 14. Kim, H.J., et al., Evaluation of Protein Expression in Housekeeping Genes across Multiple Tissues in Rats. Korean J Pathol, 2014. 48(3): p. 193-200.
13. 15. Melgar-Rojas, P., et al., Validation of Reference Genes for RT-qPCR Analysis in Noise-Induced Hearing Loss: A Study in Wistar Rat. PLoS One, 2015. 10(9): p. e0138027.
14. 16. Julian, G.S., et al., Analysis of the stability of housekeeping gene expression in the left cardiac ventricle of rats submitted to chronic intermittent hypoxia. J Bras Pneumol, 2016. 42(3): p. 211-4.
15. 17. Kirschneck, C., et al., Reference genes for valid gene expression studies on rat dental, periodontal and alveolar bone tissue by means of RT-qPCR with a focus on orthodontic tooth movement and periodontitis. Ann Anat, 2016. 204: p. 93-105.
16. 18. Benak, D., et al., Selection of optimal reference genes for gene expression studies in chronically hypoxic rat heart. Mol Cell Biochem, 2019. 461(1-2): p. 15-22.
17. 19. Kosuth, J., et al., Selection of Reliable Reference Genes for Analysis of Gene Expression in Spinal Cord during Rat Postnatal Development and after Injury. Brain Sci, 2019. 10(1).
18. 20. Han, H., et al., The optimal compound reference genes for qRT-PCR analysis in the developing rat long bones under physiological conditions and prenatal dexamethasone exposure model. Reprod Toxicol, 2020. 98: p. 242-251.
19. 21. Fiddler, J.L. and S.L. Clarke, Evaluation of candidate reference genes for quantitative real-time PCR analysis in a male rat model of dietary iron deficiency. Genes Nutr, 2021. 16(1): p. 17.
20. 22. Dragon, A.H., et al., Systematic Identification of the Optimal Housekeeping Genes for Accurate Transcriptomic and Proteomic Profiling of Tissues following Complex Traumatic Injury. Methods Protoc, 2023. 6(2).
21. 23. Sullivan-Gunn, M., et al., Choosing a stable housekeeping gene and protein is essential in generating valid gene and protein expression results. Br J Cancer, 2011. 104(6): p. 1055; author reply 1056.
22. 24. Livak, K.J. and T.D. Schmittgen, Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T)) Method. Methods, 2001. 25(4): p. 402-8.
23. 25. Hounkpe, B.W., et al., HRT Atlas v1.0 database: redefining human and mouse housekeeping genes and candidate reference transcripts by mining massive RNA-seq datasets. Nucleic Acids Research, 2020. 49(D1): p. D947-D955.