Beta-actin cannot be used as a control for gene expression in ovine interstitial cells derived from heart valves.
OBJECTIVE
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, beta-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 beta-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
beta-Actin expression in the aortic valve was significantly lower than in other valves. The mRNA level of beta-actin was four-fold lower in freshly isolated IC than in cultured IC. Once up-regulated by in-vitro culturing conditions, beta-actin expression did not change from P0 to P5. An important increase in the variation of beta-actin expression was observed in cultured cells as compared to fresh cells. Different serum conditions did not lead to different beta-actin levels.


CONCLUSIONS
Due to the variation in expression, beta-actin cannot be used as a reference for gene expression of ovine-derived heart valve IC in culture.
Cardiovascular Research Laboratory, Centrum voor Experimentele Heelkunde en Anaesthesiologie, Katholieke Universiteit Leuven, Leuven, Belgium.
Jessa
J
Yperman
Geofrey
G
De Visscher
Paul
P
Holvoet
Willem
W
Flameng
D000199:Actins / Q000235:genetics*; D000818:Animals; D002478:Cells, Cultured; D015870:Gene Expression / Q000235:genetics; D005786:Gene Expression Regulation / Q000235:genetics*; D006351:Heart Valves / Q000166:cytology*; D006801:Humans; D023421:Models, Animal; D012333:RNA, Messenger; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods; D012756:Sheep
D000199:Actins; D012333:RNA, Messenger