Reference Gene Validation for RT-qPCR in PBMCs from Asthmatic Patients with or without Obesity.

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.