The Role of Reference Gene Differences in Synchronization Control of Circadian Rhythm-Induced Gene Expression in the HT29 Cell Line

Yıl 2019, Cilt: 12 Sayı: 3, 1370 - 1380, 31.12.2019

Beyza Göncü Dilek Öztürk

https://doi.org/10.18185/erzifbed.523088

Öz

Circadian timing system controls many important vital functions through molecular clocks in mammalian organs and cells. The role of circadian rhythm in diseases has been better understood in recent years. In cell culture studies to be carried out in this area, primarily important things are ensuring synchronization and its control. In this study, the synchronization was controlled by PER2 clock gene and the two reference genes in nutrient-mediated synchronized HT-29 cells. Our aim is to investigate the role of reference genes in synchronization control. For this purpose, following serum shock HT-29 cells were harvested at 6 different times, RNA isolation was made and  PER2, ACTB, and RPLP0 gene expressions were quantified by real-time PCR (RT-PCR) assay. The results were evaluated with Fourier transformation and Linear Cosinor analysis. When the ACTB reference gene was selected, 24 and 30 hour periods showed a significant change by Fourier analysis and 24-hour period showed a significant change with Cosinor analysis. When the RPLP0 reference gene was selected, the 18 hour cycle time was significant with both analyzes. As the findings show, it is important to evaluate several parameters such as reference gene and analysis method in the initiation and control of synchronization. It is thought that this study will create awareness for in vitro studies in this area.

Anahtar Kelimeler

Synchronization, Circadien rthym, HT29, PER2

HT29 Hücre Hattında Sirkadiyen Ritme Bağlı Gen İfadesinin Kontrolünde Referans Gen Farklılığının Senkronizasyondaki Rolü

Öz

Sirkadiyen
zamanlama sistemi, memeli organ ve hücrelerinde bulunan moleküler saatler
aracılığı ile pek çok hayati fonksiyonu kontrol etmektedir. Son yıllarda
yapılan çalışmalar ile sirkadiyen ritmin hastalıklardaki rolü daha iyi
anlaşılmıştır. Bu kapsamda gerçekleştirilecek hücre kültürü çalışmalarında
öncelikli olarak senkronizasyonun sağlanması ve kontrolü önem arz etmektedir.
Sunduğumuz çalışmada besin aracılığı ile senkronize edilen HT-29 hücrelerinde,
senkronizasyon saat geni olarak da bilinen PER2
ve iki farklı referans gen ile kontrol edilmiştir. Amacımız senkronizasyon
kontrolünde referans genlerin rolünü incelemektir. Bu amaçla; serum şokunu
takiben HT-29 hücreleri T0 zamanından itibaren 6 farklı zamanda toplanarak RNA
izole edilmiş ve PER2, ACTB ve RPLP0 gen ekspresyonları gerçek zamanlı PZR (RT-PZR) deneyi ile
kantifiye edilmiştir. Sonuçlar Fourier transformasyon ve Lineer Cosinor
analizleri ile değerlendirilmiştir. ACTB
referans geni seçildiğinde Fourier transformasyon analizi ile 24 ve 30 saatlik
periyotlar anlamlı değişim gösterirken, Lineer Cosinor analizi ile sadece 24
saatlik periyot süresi anlamlı değişim göstermiştir. RPLP0 referans geni seçildiğinde her iki analiz ile de 18 saatlik
periyot süresi anlamlılık göstermiştir. Bulguların gösterdiği gibi
senkronizasyonun başlatılması ve kontrolünde referans gen ve analiz yöntemi
gibi pek çok parametrenin birlikte değerlendirilmesi önem arz etmektedir.
Yapılan çalışmanın bu alanda yapılacak in vitro çalışmalar için
farkındalık yaratacağı düşünülmektedir. 

Anahtar Kelimeler

Senkronizasyon, Sirkadiyen ritim, HT29, PER2

Kaynakça

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