Identification of reference genes for qPCR analysis during hASC long culture maintenance

PLoS One. 2017 Feb 9;12(2):e0170918. doi: 10.1371/journal.pone.0170918. eCollection 2017.


Up to now quantitative PCR based assay is the most common method for characterizing or confirming gene expression patterns and comparing mRNA levels in different sample populations. Since this technique is relative easy and low cost compared to other methods of characterization, e.g. flow cytometry, we used it to typify human adipose-derived stem cells (hASCs). hASCs possess several characteristics that make them attractive for scientific research and clinical applications. Accurate normalization of gene expression relies on good selection of reference genes and the best way to choose them appropriately is to follow the common rule of the "Best 3", at least three reference genes, three different validation software and three sample replicates. Analysis was performed on hASCs cultivated until the eleventh cell confluence using twelve candidate reference genes, initially selected from literature, whose stability was evaluated by the algorithms NormFinder, BestKeeper, RefFinder and IdealRef, a home-made version of GeNorm. The best gene panel (RPL13A, RPS18, GAPDH, B2M, PPIA and ACTB), determined in one patient by IdealRef calculation, was then investigated in other four donors. Although patients demonstrated a certain gene expression variability, we can assert that ACTB is the most unreliable gene whereas ribosomal proteins (RPL13A and RPS18) show minor inconstancy in their mRNA expression. This work underlines the importance of validating reference genes before conducting each experiment and proposes a free software as alternative to those existing.

MeSH terms

  • Actins / genetics
  • Actins / metabolism
  • Adipose Tissue / cytology
  • Adult
  • Cell Culture Techniques / methods
  • Cells, Cultured
  • Female
  • Humans
  • Mesenchymal Stem Cells / metabolism*
  • Middle Aged
  • RNA, Messenger / genetics
  • RNA, Messenger / metabolism
  • RNA, Messenger / standards*
  • Real-Time Polymerase Chain Reaction / standards*
  • Reference Standards


  • Actins
  • RNA, Messenger

Grants and funding

I state that, all the funding or sources of support received during this specific study (Fondazione Cariplo, award number 2013-1052 to RG) had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Furthermore, I also state that any individuals employed or contracted by the funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript; moreover, none of the authors has been directly payed by the funder, however, CP was the recipient of a fellowship granted by Fondazione Cariplo.