title: Identification of suitable reference genes for quantitative RT-PCR during 3T3-L1 adipocyte differentiation.
abstract: Quantitative reverse transcription PCR (qRT-PCR) is becoming increasingly important in the effort to gain insight into the molecular mechanisms underlying adipogenesis. However, the expression profile of a target gene may be misinterpreted due to the unstable expression of the reference genes under different experimental conditions. Therefore, in this study, we investigated the expression stability of 10 commonly used reference genes during 3T3-L1 adipocyte differentiation. The mRNA expression levels of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and transferrin receptor (TFRC) significantly increased during the course of 3T3-L1 adipocyte differentiation, which was decreased by berberine, an inhibitor of adipogenesis. Three popular algorithms, GeNorm, NormFinder and BestKeeper, identified 18 ribosomal RNA and hydroxymethylbilane synthase (HMBS) as the most stable reference genes, while GAPDH and TFRC were the least stable ones. Peptidylprolyl isomerase A [PIPA (cyclophilin A)], ribosomal protein, large, P0 (36-B4), beta-2-microglobulin (B2M), α1-tubulin, hypoxanthine-guanine phosphoribosyltransferase (HPRT) and β-actin showed relatively stable expression levels. The choice of reference genes with various expression stabilities exerted a profound influence on the expression profiles of 2 target genes, peroxisome proliferator-activated receptor (PPAR)γ2 and C/EBPα. In addition, western blot analysis revealed that the increased protein expression of GAPDH was markedly inhibited by berberine during adipocyte differentiation. This study highlights the importance of selecting suitable reference genes for qRT-PCR studies of gene expression during the process of adipogenesis.
authors.affiliation: Shanghai Institute of Endocrine and Metabolic Diseases, Department of Endocrine and Metabolic Diseases, Shanghai Clinical Center for Endocrine and Metabolic Diseases, Ruijin Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai 200025, P.R. China.
authors.forename: Juan
authors.initials: J
authors.lastname: Zhang
authors.affiliation: Shanghai Institute of Endocrine and Metabolic Diseases, Department of Endocrine and Metabolic Diseases, Shanghai Clinical Center for Endocrine and Metabolic Diseases, Ruijin Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai 200025, P.R. China.
authors.forename: Hongju
authors.initials: H
authors.lastname: Tang
authors.affiliation: Shanghai Institute of Endocrine and Metabolic Diseases, Department of Endocrine and Metabolic Diseases, Shanghai Clinical Center for Endocrine and Metabolic Diseases, Ruijin Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai 200025, P.R. China.
authors.forename: Yuqing
authors.initials: Y
authors.lastname: Zhang
authors.affiliation: Shanghai Institute of Endocrine and Metabolic Diseases, Department of Endocrine and Metabolic Diseases, Shanghai Clinical Center for Endocrine and Metabolic Diseases, Ruijin Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai 200025, P.R. China.
authors.forename: Ruyuan
authors.initials: R
authors.lastname: Deng
authors.affiliation: Department of Geratology, East Hospital, Shanghai Tongji University, Shanghai 200120, P.R. China.
authors.forename: Li
authors.initials: L
authors.lastname: Shao
authors.affiliation: Shanghai Institute of Endocrine and Metabolic Diseases, Department of Endocrine and Metabolic Diseases, Shanghai Clinical Center for Endocrine and Metabolic Diseases, Ruijin Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai 200025, P.R. China.
authors.forename: Yun
authors.initials: Y
authors.lastname: Liu
authors.affiliation: Shanghai Institute of Endocrine and Metabolic Diseases, Department of Endocrine and Metabolic Diseases, Shanghai Clinical Center for Endocrine and Metabolic Diseases, Ruijin Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai 200025, P.R. China.
authors.forename: Fengying
authors.initials: F
authors.lastname: Li
authors.affiliation: Shanghai Institute of Endocrine and Metabolic Diseases, Department of Endocrine and Metabolic Diseases, Shanghai Clinical Center for Endocrine and Metabolic Diseases, Ruijin Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai 200025, P.R. China.
authors.forename: Xiao
authors.initials: X
authors.lastname: Wang
authors.affiliation: Shanghai Institute of Endocrine and Metabolic Diseases, Department of Endocrine and Metabolic Diseases, Shanghai Clinical Center for Endocrine and Metabolic Diseases, Ruijin Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai 200025, P.R. China.
authors.forename: Libin
authors.initials: L
authors.lastname: Zhou
mesh_terms: D041721:3T3-L1 Cells; D000199:Actins / Q000235:genetics; D017667:Adipocytes / Q000166:cytology* / Q000378:metabolism*; D000465:Algorithms; D000818:Animals; D015703:Antigens, CD / Q000235:genetics; D002454:Cell Differentiation / Q000235:genetics*; D024581:Glyceraldehyde-3-Phosphate Dehydrogenase (Phosphorylating) / Q000235:genetics; D011163:Hydroxymethylbilane Synthase / Q000235:genetics; D007041:Hypoxanthine Phosphoribosyltransferase / Q000235:genetics; D051379:Mice; D012335:RNA, Ribosomal / Q000235:genetics; D011990:Receptors, Transferrin / Q000235:genetics; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods*
chemical_list: D000199:Actins; D015703:Antigens, CD; C068126:CD71 antigen; D012335:RNA, Ribosomal; D011990:Receptors, Transferrin; D024581:Glyceraldehyde-3-Phosphate Dehydrogenase (Phosphorylating); D007041:Hypoxanthine Phosphoribosyltransferase; D011163:Hydroxymethylbilane Synthase