Abstract
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.
Highlights
Stem cells are unspecialized cells that possess three basic characteristics: self-renewal, long life and ability to differentiate into various cell lines
After extraction from adipose tissue of healthy donor, human adipose-derived stem cells (hASCs) were characterized by flow cytometry and the results have been reported in a previous paper [26]
NormFinder algorithm assigns a lower value for higher stable gene and indicates that, in our experimental conditions, the three most stable genes are RPL13A, RPS18 and glyceraldeihyde-3-phospate dehydrogenase (GAPDH)
Summary
Stem cells are unspecialized cells that possess three basic characteristics: self-renewal, long life and ability to differentiate into various cell lines. Human adult somatic stem cells are present throughout all the body including adipose tissue [1,2]. Later studies showed the potential ability of adipose stem cells (hASCs) to differentiate towards different cell lineages [3,4,5,6,7,8] and it was recently suggested that the hASCs can be pluripotent. HASCs are very attractive for scientific research and clinical applications as they can be extracted and cultivated in vitro, have a greater differentiation potential than other mesenchymal stem cells and PLOS ONE | DOI:10.1371/journal.pone.0170918. Later studies showed the potential ability of adipose stem cells (hASCs) to differentiate towards different cell lineages [3,4,5,6,7,8] and it was recently suggested that the hASCs can be pluripotent. hASCs are very attractive for scientific research and clinical applications as they can be extracted and cultivated in vitro, have a greater differentiation potential than other mesenchymal stem cells and PLOS ONE | DOI:10.1371/journal.pone.0170918 February 9, 2017
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