Gentile Adriana-Mariel, Lhamyani Said, Coín-Aragüez Leticia, Oliva-Olivera Wilfredo, Zayed Hatem, Vega-Rioja Antonio, Monteseirin Javier, Romero-Zerbo Silvana-Yanina, Tinahones Francisco-José, Bermúdez-Silva Francisco-Javier, El Bekay Rajaa
IBIMA, Universidad de Málaga, Campus Teatinos s/n, 29010, Málaga, España.
CIBER Pathophysiology of obesity and nutrition CB06/03, Carlos III Health Institute, Malaga, 29010, Spain, Laboratory of Biomedical Research, Virgen de la Victoria Clinical University Hospital, Málaga, 29010, Spain.
PLoS One. 2016 Jun 15;11(6):e0157002. doi: 10.1371/journal.pone.0157002. eCollection 2016.
Real-time or quantitative PCR (qPCR) is a useful technique that requires reliable reference genes for data normalization in gene expression analysis. Adipogenesis is among the biological processes suitable for this technique. The selection of adequate reference genes is essential for qPCR gene expression analysis of human Vascular Stromal Cells (hVSCs) during their differentiation into adipocytes. To the best of our knowledge, there are no studies validating reference genes for the analyses of visceral and subcutaneous adipose tissue hVSCs from subjects with different Body Mass Index (BMI) and Homeostatic Model Assessment of Insulin Resistance (HOMA-IR) index. The present study was undertaken to analyze this question. We first analyzed the stability of expression of five potential reference genes: CYC, GAPDH, RPL13A, EEF1A1, and 18S ribosomal RNA, during in vitro adipogenic differentiation, in samples from these types of patients. The expression of RPL13A and EEF1A1 was not affected by differentiation, thus being these genes the most stable candidates, while CYC, GAPDH, and 18S were not suitable for this sort of analysis. This work highlights that RPL13A and EEF1A1 are good candidates as reference genes for qPCR analysis of hVSCs differentiation into adipocytes from subjects with different BMI and HOMA-IR.
实时定量聚合酶链反应（qPCR）是一种有用的技术，在基因表达分析中需要可靠的内参基因进行数据标准化。脂肪生成是适合该技术的生物学过程之一。选择合适的内参基因对于人血管基质细胞（hVSCs）向脂肪细胞分化过程中的qPCR基因表达分析至关重要。据我们所知，尚无研究验证不同体重指数（BMI）和胰岛素抵抗稳态模型评估（HOMA-IR）指数的受试者内脏和皮下脂肪组织hVSCs分析的内参基因。本研究旨在分析这个问题。我们首先分析了五种潜在内参基因：CYC、GAPDH、RPL13A、EEF1A1和18S核糖体RNA在这些类型患者样本体外脂肪生成分化过程中的表达稳定性。RPL13A和EEF1A1的表达不受分化影响，因此是最稳定的候选基因，而CYC、GAPDH和18S不适合此类分析。这项工作突出表明，RPL13A和EEF1A1是不同BMI和HOMA-IR受试者hVSCs向脂肪细胞分化的qPCR分析中作为内参基因的良好候选者。
