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

ABSTRACT: 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 ? smooth muscle actin (?-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 ?-SMA and ß-actin from other actin isoforms. RESULTS:Real-time PCRs using self-designed mouse, human and rat specific ?-SMA or ß-actin primer pairs resulted in the specific amplification of the artificial DNA templates corresponding to mouse, human or rat ?-SMA or ß-actin, however ß-actin showed cross-reaction with the housekeeping ?-cyto-actin. We have shown that the use of improperly designed literary primer pairs significantly affects the results of PCRs measuring mRNA expression of ?-SMA or ß-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 ?-SMA and ß-actin isoforms. We demonstrated the importance of primer specificity in experiments where the results are normalized to the expression of ß-actin especially when fibrosis and thus increased expression of ?-SMA is occur.

SUBMITTER: Veres-Szekely A 

PROVIDER: S-EPMC5408491 | biostudies-literature | 2017 Apr

REPOSITORIES: biostudies-literature