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Updated: Aug 21, 2026
Protein Isolation from the Developing Embryonic Mouse Heart Valve Region
Published on: September 23, 2014
Beta-actin cannot be used as a control for gene expression in ovine interstitial cells derived from heart valves
Jessa Yperman1, Geofrey De Visscher, Paul Holvoet
1Cardiovascular Research Laboratory, Centrum voor Experimentele Heelkunde en Anaesthesiologie, Katholieke Universiteit Leuven, Leuven, Belgium.
Insights
Beta-actin is not a reliable housekeeping gene for ovine heart valve interstitial cells in culture. Its expression varies significantly between fresh and cultured cells and across different valves.
Area of Science:
- Cardiovascular Biology
- Molecular Biology
- Gene Expression Analysis
Background:
- Housekeeping genes are essential for normalizing gene expression data in studies.
- Beta-actin is a commonly used housekeeping gene in various tissues.
Purpose of the Study:
- To evaluate the suitability of beta-actin as a housekeeping gene in ovine heart valve interstitial cells.
- To assess beta-actin expression in fresh tissue versus cultured cells and across different serum conditions.
Main Methods:
- Isolation of interstitial cells (IC) from ovine heart valves using outgrowth explant method.
- Culture of IC under varying serum conditions up to passage 5.
- Quantification of beta-actin mRNA expression via reverse transcription-polymerase chain reaction (RT-PCR).
Main Results:
- Beta-actin expression was significantly lower in the aortic valve compared to other valves.
- Cultured IC showed a four-fold higher beta-actin mRNA level than freshly isolated IC.
- While stable from passage 0 to 5, beta-actin expression exhibited increased variation in cultured cells.
Conclusions:
- Beta-actin demonstrates significant variability in expression within ovine heart valve interstitial cells.
- The use of beta-actin as a reference gene for gene expression studies in cultured ovine heart valve IC is not recommended.
- Further validation of reference genes is crucial for accurate gene expression analysis in this cell type.
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, 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.
Conclusion:
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.
