Romaniuk-Drapała Aleksandra, Kosicka-Noworzyń Katarzyna, Sheng Yi-Hua, Yohn Christine, Brunetti Luigi, Kagan Leonid
Department of Clinical Chemistry and Molecular Diagnostics, Poznan University of Medical Sciences, 3 Rokietnicka Street, Poznan, 60-806, Poland.
Department of Pharmaceutics, Ernest Mario School of Pharmacy, Rutgers, The State University of New Jersey, 160 Frelinghuysen Road, Piscataway, NJ, 08854, USA.
Sci Rep. 2025 Feb 13;15(1):5347. doi: 10.1038/s41598-025-87911-x.
Given the obesity epidemic and the prevalence of comorbidities, there is an ongoing need to understand the health consequences of this disease state better. Understanding gene expression signals will facilitate the identification of mechanisms of kidney and liver dysfunction/disease often present in individuals with obesity. qPCR is the standard method for studying changes in relative gene expression. Reference genes (RGs) are obligatory for accurately normalizing mRNA transcript levels across samples. Despite the prevalence of qPCR, the reliability of the data is often compromised because RGs are still used without validation or have proven to be unstable in different tissues and various diseases. In this study, we validated seven reference genes (ACTB, B2M, RPLP0, HPRT1, GAPDH, 18S rRNA, and PPIA) using human liver tissue from 15 lean individuals and 17 individuals with a BMI ≥ 25 and human kidney tissue from 13 lean individuals and 15 individuals with a BMI ≥ 25. Cross-validation of expression stability was performed using the RefFinder platform with four algorithms: NormFinder, BestKeeper, geNorm, and the comparative ΔCt method. In obesity-related studies, the most suitable reference genes in gene expression studies are RPLP0 and HPRT1 in human kidney tissue and RPLP0 and GAPDH in the liver.
鉴于肥胖症流行以及合并症的普遍存在，人们一直需要更好地了解这种疾病状态对健康的影响。了解基因表达信号将有助于识别肥胖个体中常见的肾和肝功能障碍/疾病的机制。定量聚合酶链反应（qPCR）是研究相对基因表达变化的标准方法。参考基因（RGs）对于准确标准化样本间的mRNA转录水平至关重要。尽管qPCR应用广泛，但数据的可靠性常常受到影响，因为参考基因仍在未经验证的情况下使用，或者已被证明在不同组织和各种疾病中不稳定。在本研究中，我们使用了来自15名体重正常个体和17名体重指数（BMI）≥25的个体的人肝组织，以及来自13名体重正常个体和15名BMI≥25的个体的人肾组织，对七个参考基因（ACTB、B2M、RPLP0、HPRT1、GAPDH、18S rRNA和PPIA）进行了验证。使用RefFinder平台通过四种算法（NormFinder、BestKeeper、geNorm和比较ΔCt法）对表达稳定性进行交叉验证。在肥胖相关研究中，基因表达研究中最合适的参考基因在人肾组织中是RPLP0和HPRT1，在肝脏中是RPLP0和GAPDH。
