Beta-Actin Cannot be Used as a Control for Gene Expression in Ovine Interstitial Cells Derived from Heart Valves
Jessa Yperman, Geofrey De Visscher, Paul Holvoet, Willem Flameng Cardiovascular Research Laboratory, Centrum voor Experimentele Heelkunde en Anaesthesiologie, Katholieke Universiteit Leuven, Leuven, Belgium
Background and aim of the study: In gene expression studies, endogenous controls that are constitutively expressed (housekeeping genes) are commonly used to normalize for variations in cDNA synthesis efficiency. In the present study, a frequently used control gene, b-actin, was examined in ovine heart valves to evaluate its applicability as a housekeeping gene for this tissue. Methods: Interstitial cells (IC) of the four heart valves were isolated using the outgrowth explant method. Cells were cultured under different serum conditions (10% or 20% fetal bovine serum or 20% sheep serum) up to passage (P) 5. mRNA from fresh tissue and from cells at P0 and P5 was isolated, and expression of b-actin determined using reverse transcription-polymerase chain reaction (RT-PCR). An identical control
sample was used for each PCR and each gel electrophoresis. Data were expressed as a relative value of this control sample. Results: b-Actin expression in the aortic valve was significantly lower than in other valves. The mRNA level of b-actin was four-fold lower in freshly isolated IC than in cultured IC. Once up-regulated by in-vitro culturing conditions, b-actin expression did not change from P0 to P5. An important increase in the variation of b-actin expression was observed in cultured cells as compared to fresh cells. Different serum conditions did not lead to different b-actin levels. Conclusion: Due to the variation in expression, b-actin cannot be used as a reference for gene expression of ovine-derived heart valve IC in culture. The Journal of Heart Valve Disease 2004;13:848-853
Click the above hyperlink to view the article, right click (Ctrl click on a Mac) to open in a new browser window or tab.
Purchase this Article
Please click the button below to purchase this article. Single article purchases are provided at $50.00 per article. Upon clicking the button below, single article user account subscription details are requested and, upon successful payment, a single article user account is created. Single articles are availble in your account for seven days after purchase.