Selective measurement of a smooth muscle actin: why β-actin can not be used as a housekeeping gene when tissue fibrosis occurs

Background: Prevalence of fibroproliferative diseases, including chronic kidney disease is rapidly increasing and has become a major public health problem worldwide. Fibroproliferative diseases are characterized by increased expression of a smooth muscle actin (alpha-SMA) that belongs to the family of the six conserved actin isoforms showing high degree homology. The aim of the present study was to develop real-time PCRs that clearly discriminate alpha-SMA and beta-actin from other actin isoforms. Results: Real-time PCRs using self-designed mouse, human and rat specific alpha-SMA or beta-actin primer pairs resulted in the specific amplification of the artificial DNA templates corresponding to mouse, human or rat alpha-SMA or beta-actin, however beta-actin showed cross-reaction with the housekeeping gamma-cyto-actin. We have shown that the use of improperly designed literary primer pairs significantly affects the results of PCRs measuring mRNA expression of alpha-SMA or beta-actin in the kidney of mice underwent UUO. Conclusion: We developed a set of carefully designed primer pairs and PCR conditions to selectively determine the expression of mouse, human or rat alpha-SMA and beta-actin isoforms. We demonstrated the importance of primer specificity in experiments where the results are normalized to the expression of beta-actin especially when fibrosis and thus increased expression of alpha-SMA is occur.