Identification of suitable reference genes for quantitative gene expression analysis in rat adipose stromal cells induced to trilineage 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 28day treatments (ALL28); Ppia and Hprt1 (adipogenic differentiation; A28), B2m (chondrogenic differentiation; C28), Rpl5 (controls maintained in complete culture medium; CCM), Rplp0 (osteogenic differentiation for 3days; O3), Rpl13a and Actb (osteogenic differentiation for 7days; O7), Rplp0 and Ppia (osteogenic differentiation for 14days; O14), Hprt1 and Ppia (osteogenic differentiation for 28days; 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.
Laboratory of Stem Cells and Tissue Engineering, Lutheran University of Brazil, Canoas 92425-900, RS, Brazil. Electronic address: brnpaivas@gmail.com.
Bruno Paiva Dos
BP
Santos
Laboratory of Stem Cells and Tissue Engineering, Lutheran University of Brazil, Canoas 92425-900, RS, Brazil. Electronic address: lu.lucianafcosta@gmail.com.
Luciana Fraga
LF
da Costa Diesel
Laboratory of Stem Cells and Tissue Engineering, Lutheran University of Brazil, Canoas 92425-900, RS, Brazil. Electronic address: lindolfomeirelles@gmail.com.
Lindolfo
L
da Silva Meirelles
Laboratory of Stem Cells and Tissue Engineering, Lutheran University of Brazil, Canoas 92425-900, RS, Brazil. Electronic address: nance.nardi@ulbra.br.
Nance Beyer
NB
Nardi
Laboratory of Stem Cells and Tissue Engineering, Lutheran University of Brazil, Canoas 92425-900, RS, Brazil. Electronic address: melissa.camassola@ulbra.br.
Melissa
M
Camassola
D050156:Adipogenesis; D000273:Adipose Tissue / Q000166:cytology / Q000378:metabolism*; D000465:Algorithms; D000818:Animals; D000943:Antigens, Differentiation / Q000096:biosynthesis* / Q000235:genetics; D002454:Cell Differentiation; D002478:Cells, Cultured; D020219:Chondrogenesis; D020869:Gene Expression Profiling / Q000379:methods / Q000592:standards*; D005786:Gene Expression Regulation; D010012:Osteogenesis; D051381:Rats; D017154:Stromal Cells / Q000166:cytology / Q000378:metabolism
D000943:Antigens, Differentiation
Adipose stem cell; Endogenous control; Gene expression; Housekeeping gene; Internal control; Normalization; RT-qPCR