Department of Biochemistry and Molecular Biology, Faculty of Medicine, Dalhousie University, Dalhousie Medicine New Brunswick, 100 Tucker Park Road, Saint John, E2L 4L5, New Brunswick, Canada.
Deparment of Medicine, Faculty of Medicine, Dalhousie University, Dalhousie Medicine New Brunswick, 100 Tucker Park Road, Saint John, E2L 4L5, New Brunswick, Canada.
Sci Rep. 2017 Jun 15;7(1):3612. doi: 10.1038/s41598-017-03730-9.
The global incidence of obesity has led to an increasing need for understanding the molecular mechanisms that drive this epidemic and its comorbidities. Quantitative real-time RT-PCR (RT-qPCR) is the most reliable and widely used method for gene expression analysis. The selection of suitable reference genes (RGs) is critical for obtaining accurate gene expression information. The current study aimed to identify optimal RGs to perform quantitative transcriptomic analysis based on RT-qPCR for obesity and diabetes research, employing in vitro and mouse models, and human tissue samples. Using the ReFinder program we evaluated the stability of a total of 15 RGs. The impact of choosing the most suitable RGs versus less suitable RGs on RT-qPCR results was assessed. Optimal RGs differed between tissue and cell type, species, and experimental conditions. By employing different sets of RGs to normalize the mRNA expression of peroxisome proliferator-activated receptor gamma coactivator 1-alpha (PGC1α), we show that sub-optimal RGs can markedly alter the PGC1α gene expression profile. Our study demonstrates the importance of validating RGs prior to normalizing transcriptional expression levels of target genes and identifies optimal RG pairs for reliable RT-qPCR normalization in cells and in human and murine muscle and adipose tissue for obesity/diabetes research.
全球肥胖症的发病率不断上升，这使得人们越来越需要了解推动这一流行病及其合并症的分子机制。实时荧光定量 RT-PCR（RT-qPCR）是分析基因表达最可靠和最常用的方法。选择合适的内参基因（RGs）对于获得准确的基因表达信息至关重要。本研究旨在确定基于 RT-qPCR 的肥胖和糖尿病研究的最佳内参基因，使用体外和小鼠模型以及人体组织样本。我们使用 ReFinder 程序评估了总共 15 个 RG 的稳定性。评估了选择最合适的 RG 与不太合适的 RG 对 RT-qPCR 结果的影响。最佳 RG 在组织和细胞类型、物种和实验条件之间存在差异。通过使用不同的 RG 集来归一化过氧化物酶体增殖物激活受体γ共激活因子 1-α（PGC1α）的 mRNA 表达，我们表明，次优 RG 会显著改变 PGC1α 基因表达谱。我们的研究表明，在对靶基因的转录表达水平进行归一化之前，验证 RG 非常重要，并确定了肥胖/糖尿病研究中用于细胞和人体及鼠类肌肉和脂肪组织中可靠 RT-qPCR 归一化的最佳 RG 对。
