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