Research paperIdentification of suitable reference genes for quantitative gene expression analysis in rat adipose stromal cells induced to trilineage differentiation

•There is no reference gene suitable for all conditions and time points analyzed.•Different rankings of stable genes were observed according to differentiation.•We set information for normalization of gene expression in rASC differentiation.•The more heterogenic the sample is, the more reference genes are needed.•Different reference genes are stable along osteogenic differentiation.

This study was designed to (i) identify stable reference genes for the analysis of gene expression during in vitro differentiation of rat adipose stromal cells (rASCs), (ii) recommend stable genes for individual treatment conditions, and (iii) validate these genes by comparison with normalization results from stable and unstable reference genes. On the basis of a literature review, eight genes were selected: Actb, B2m, Hprt1, Ppia, Rplp0, Rpl13a, Rpl5, and Ywhaz. Genes were ranked according to their stability under different culture conditions as assessed using GenNorm, NormFinder, and RefFinder algorithms. Although the employed algorithms returned different rankings, the most frequently top-ranked genes were: B2m and/or Ppia for all 28 day treatments (ALL28); Ppia and Hprt1 (adipogenic differentiation; A28), B2m (chondrogenic differentiation; C28), Rpl5 (controls maintained in complete culture medium; CCM), Rplp0 (osteogenic differentiation for 3 days; O3), Rpl13a and Actb (osteogenic differentiation for 7 days; O7), Rplp0 and Ppia (osteogenic differentiation for 14 days; O14), Hprt1 and Ppia (osteogenic differentiation for 28 days; O28), as well as Actb (all osteogenesis time points combined; ALLOSTEO). The obtained results indicate that the performance of reference genes depends on the differentiation protocol and on the analysis time, thus providing valuable information for the design of RT-PCR experiments.