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.
Department of Geratology, East Hospital, Shanghai Tongji University, Shanghai 200120, P.R. China.
Int J Mol Med. 2014 May;33(5):1209-18. doi: 10.3892/ijmm.2014.1695. Epub 2014 Mar 11.
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.
定量逆转录聚合酶链反应（qRT-PCR）在深入了解脂肪生成的分子机制方面变得越来越重要。然而，由于目标基因在不同实验条件下的参考基因表达不稳定，其表达谱可能会被误解。因此，在这项研究中，我们研究了 10 个常用参考基因在 3T3-L1 脂肪细胞分化过程中的表达稳定性。甘油醛-3-磷酸脱氢酶（GAPDH）和转铁蛋白受体（TFRC）的 mRNA 表达水平在 3T3-L1 脂肪细胞分化过程中显著增加，而脂肪生成抑制剂小檗碱则降低了其表达。GeNorm、NormFinder 和 BestKeeper 这 3 种流行算法确定了 18S 核糖体 RNA 和羟甲基胆素合酶（HMBS）作为最稳定的参考基因，而 GAPDH 和 TFRC 则是最不稳定的参考基因。肽基脯氨酰顺反异构酶 A[PIPA（环孢素 A）]、核糖体蛋白、大、P0（36-B4）、β-2-微球蛋白（B2M）、α1-微管蛋白、次黄嘌呤-鸟嘌呤磷酸核糖基转移酶（HPRT）和β-肌动蛋白的表达水平相对稳定。具有不同表达稳定性的参考基因的选择对 2 个靶基因（过氧化物酶体增殖物激活受体γ2 和 C/EBPα）的表达谱产生了深远的影响。此外，Western blot 分析显示，在脂肪细胞分化过程中，小檗碱显著抑制了 GAPDH 蛋白表达的增加。这项研究强调了在脂肪生成过程中选择合适的参考基因进行 qRT-PCR 研究基因表达的重要性。
