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

Apor Veres-Székely, Domonkos Pap, Erna Sziksz, Eszter Jávorszky, Réka Rokonay, Rita Lippai, Kálmán Tory, Andrea Fekete, Tivadar Tulassay, Attila J. Szabó, Ádám Vannay

Research output: Contribution to journalArticle

10 Citations (Scopus)

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 α smooth muscle actin (α-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 α-SMA and ß-actin from other actin isoforms. Results: Real-time PCRs using self-designed mouse, human and rat specific α-SMA or ß-actin primer pairs resulted in the specific amplification of the artificial DNA templates corresponding to mouse, human or rat α-SMA or ß-actin, however ß-actin showed cross-reaction with the housekeeping γ-cyto-actin. We have shown that the use of improperly designed literary primer pairs significantly affects the results of PCRs measuring mRNA expression of α-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 α-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 α-SMA is occur.

Original languageEnglish
Article number12
JournalBMC Molecular Biology
Volume18
Issue number1
DOIs
Publication statusPublished - Apr 27 2017

Keywords

  • Actin
  • Fibrosis
  • Primer design
  • Real-time PCR
  • ß-actin
  • α-SMA

ASJC Scopus subject areas

  • Molecular Biology

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