Bantulà Marina, Arismendi Ebymar, Picado César, Mullol Joaquim, Roca-Ferrer Jordi, Tubita Valeria
August Pi i Sunyer Biomedical Research Institute (IDIBAPS), Hospital Clinic, Universitat de Barcelona, 08036 Barcelona, Spain.
Department of Pneumology, Hospital Clinic, 08036 Barcelona, Spain.
Methods Protoc. 2022 Apr 22;5(3):35. doi: 10.3390/mps5030035.
Obesity is known to impair the efficacy of glucocorticoid medications for asthma control. Glucocorticoid-induced gene expression studies may be useful to discriminate those obese asthmatic patients who present a poor response to glucocorticoids. The expression of genes of interest is normalized with respect to reference genes (RGs). Ideally, RGs have a stable expression in different samples and are not affected by experimental conditions. The objective of this work was to analyze suitable RGs to study the role of glucocorticoid-induced genes in obese asthmatic patients in further research. The gene expression of eight potential RGs (, , , , , , , and ) was assessed with reverse transcription-quantitative polymerase chain reaction in peripheral blood mononuclear cells (PBMCs) from asthmatic, obese asthmatic, and healthy individuals. Their stability was analyzed using four different algorithms-BestKeeper, ΔCt, geNorm, and NormFinder. geNorm analysis recommended the use of a minimum of three genes for normalization. Moreover, intergroup variation due to the treatment was calculated by NormFinder, which found that was the gene that was least affected by different treatments. Comprehensive rankings indicated and as the best RGs for qPCR in PBMCs from healthy and asthmatic subjects, while and were the best for obese asthmatic subjects. Finally, our results demonstrated that and were the most stable RGs among all groups, whereas , , and were the worst shared ones.
众所周知，肥胖会削弱糖皮质激素药物控制哮喘的疗效。糖皮质激素诱导的基因表达研究可能有助于鉴别那些对糖皮质激素反应不佳的肥胖哮喘患者。感兴趣的基因表达相对于参考基因（RGs）进行标准化。理想情况下，参考基因在不同样本中具有稳定的表达，且不受实验条件影响。本研究的目的是分析合适的参考基因，以便在进一步研究中探讨糖皮质激素诱导基因在肥胖哮喘患者中的作用。采用逆转录定量聚合酶链反应评估了哮喘患者、肥胖哮喘患者和健康个体外周血单个核细胞（PBMCs）中8个潜在参考基因（、、、、、、和）的基因表达。使用四种不同算法——BestKeeper、ΔCt、geNorm和NormFinder分析它们的稳定性。geNorm分析建议至少使用三个基因进行标准化。此外，NormFinder计算了治疗引起的组间差异，发现是受不同治疗影响最小的基因。综合排名表明，对于健康和哮喘受试者的PBMCs，和是qPCR的最佳参考基因，而对于肥胖哮喘受试者，和是最佳参考基因。最后，我们的结果表明，和是所有组中最稳定的参考基因，而、和是最不稳定的共享参考基因。
