title: Stability of Reference Genes during Tri-Lineage Differentiation of Human Adipose-Derived Stromal Cells.
abstract: Quantitative real-time PCR can detect variations in gene expression. The identification of the stable reference genes (RGs) is necessary to evaluate the expression of specific genes of interest under various conditions in many cell types, including human adipose-derived stromal cells (hASCs). In this study, we used the algorithms BestKeeper, NormFinder, geNorm, and RefFinder to investigate the stability of 15 potential RGs (B2M, eEF1A1, GAPDH, H2AFZ, HMBS, HPRT1, PGK1, PPIA, RPL5, SDHA, TBP, TKT, TRFC, TUBB, and UBC) in hASCs during control, adipo-, chondro-, and osteogenic differentiation for 28 days. RPL5, GAPDH, H2AFZ, and HPRT1 were the most stable RGs, while B2M and UBC were the least stable RGs for the majority of group analyses (tri-lineage differentiation and control analyzed combined or individually; each lineage combined with the control). These RGs were used to normalize adipo- (FABP4, LPL, and PPARG), chondro- (COMP and SOX9), and osteogenic gene expression markers (BMP4, COL1A1, and RUNX2). Each marker showed a similar expression when normalized by H2AFZ, HPRT1, or RPL5, confirming that these RGs exhibit stable expression. However, GAPDH, B2M, and UBC exhibited high standard deviation (SD), down-regulated and/or up-regulated differentiation gene expression markers when compared with stable RGs, demonstrating that these RGs are unstable.
authors.forename: Luciana Fraga da Costa
authors.initials: LF
authors.lastname: Diesel
authors.forename: Bruno Paiva
authors.initials: BP
authors.lastname: dos Santos
authors.forename: Bruno Corrêa
authors.initials: BC
authors.lastname: Bellagamba
authors.forename: Angelo Syrillo
authors.initials: AS
authors.lastname: Pretto Neto
authors.forename: Pedro Bins
authors.initials: PB
authors.lastname: Ely
authors.forename: Lindolfo da Silva
authors.initials: Lda S
authors.lastname: Meirelles
authors.forename: Nance Beyer
authors.initials: NB
authors.lastname: Nardi
authors.forename: Melissa
authors.initials: M
authors.lastname: Camassola
mesh_terms: D017667:Adipocytes / Q000166:cytology; D002454:Cell Differentiation / Q000235:genetics*; D018507:Gene Expression Regulation, Developmental; D006801:Humans; D059630:Mesenchymal Stem Cells; D010012:Osteogenesis / Q000235:genetics; D014176:Protein Biosynthesis / Q000235:genetics*