Circadian rhythm disruptions are increasingly recognized in disorders such as obstructive sleep apnea (OSA), yet analysis of 24 h gene expression patterns remains challenging due to the lack of reliable reference genes for normalization. Even commonly used housekeeping genes may exhibit circadian oscillations, which can confound rhythmic gene expression analyses and hinder biomarker identification. To address this limitation, we evaluated the gene expression stability of 11 commonly used housekeeping genes in blood collected every 6 h over 24 h period from 40 adults with varying OSA severity and controls. Stability ranking by analytical tools RefFinder and EndoGeneAnalyzer identified ACTB (β-actin) and RPL13A (ribosomal protein L13a) as the most consistent reference genes, with minimal intra- and inter-individual variability across sampling times and disease groups. Their suitability was assessed by personalized cosinor analysis of core clock genes (BMAL1, PER2, CRY1), demonstrating that appropriate normalization enables detection of circadian oscillations in clinical samples. Using the optimal normalization, CosinorPy analysis of the core clock genes revealed significant circadian oscillations of at least one clock gene in the studied participants. These findings establish ACTB and RPL13A as robust reference genes for blood-based circadian studies of OSA and provide an important methodological framework for future circadian biomarker research.

Obstructive sleep apnea (OSA), one of the most common breathing disorders during sleep, is associated with circadian rhythm disruption, yet circadian genes expressed in blood remain largely unexplored as biomarkers. Reliable circadian gene expression analysis requires stable reference genes for qRT-PCR normalization, but no clear guidance exists for blood-based circadian studies in OSA. We evaluated the expression of 11 candidate reference genes ( GAPDH, ACTB, RPL13A, PPIB, TBP, HPRT1, PPIA, SDHA, TUBB2A, UBC ) in whole blood collected every 6 hours over 24 hours from 40 participants who underwent overnight respiratory polygraphy. Gene stability was assessed with RefFinder and EndoGeneAnalyzer across OSA severity levels, intra-individual variability, and time-point variability. ACTB and RPL13A consistently emerged as the most stable reference genes under all conditions. Their robustness was confirmed by personalized cosinor analysis of core clock genes ( BMAL1, PER2, CRY1 ), showing that reliable normalization enables detection of circadian oscillations in clinical samples. While population-level analysis revealed no significant rhythmicity, individual profiles showed oscillations in 25 participants, independent of OSA severity. These findings identify ACTB and RPL13A as strong candidates for circadian transcriptomic studies in blood and provide a methodological foundation for biomarker discovery in sleep medicine, molecular medicine, and translational chronobiology.

Circadian rhythm disruptions are increasingly recognized in disorders such as obstructive sleep apnea (OSA), yet analysis of 24-hour gene expression patterns remains challenging due to the lack of reliable reference genes for normalization. Even commonly used housekeeping genes may exhibit circadian oscillations, which can confound rhythmic gene expression analyses and hinder biomarker identification. To address this limitation, we evaluated the gene expression stability of 11 commonly used housekeeping genes in blood collected every 6 hours over 24-hour period from 40 adults with varying OSA severity and controls. Stability ranking by analytical tools RefFinder and EndoGeneAnalyzer identified ACTB (β-actin) and RPL13A (ribosomal protein L13a) as the most consistent reference genes, with minimal intra- and inter-individual variability across sampling times and disease groups. Their suitability was assessed by personalized cosinor analysis of core clock genes (BMAL1, PER2, CRY1), demonstrating that appropriate normalization enables detection of circadian oscillations in clinical samples. Using the optimal normalization, CosinorPy analysis of the core clock genes revealed significant circadian oscillations of at least one clock gene in the studied participants. These findings establish ACTB and RPL13A as robust reference genes for blood-based circadian studies of OSA and provide an important methodological framework for future circadian biomarker research.