20.500.12556/RUL-184903 Reference genes for circadian profiling of core clock genes in the blood of obstructive sleep apnea patients 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. circadian rhythms obstructive sleep apnea reference genes qRT-PCR normalization gene expression stability core clock genes cirkadiani ritmi obstruktivna apneja v spanju referenčni geni normalizacija qRT-PCR stabilnost izražanja genov osrednji geni uravnavanja ritma ključni geni biološke ure true false true Angleški jezik Slovenski jezik Članek v reviji 2026-07-17 07:05:59 2026-07-17 07:06:07 2026-08-11 07:24:21 0000-00-00 00:00:00 2026 0 0 18 str. iss. 7, art. 1013 Vol. 16 2026 0000-00-00 Zaloznikova Objavljeno NiDoloceno 0000-00-00 0000-00-00 0000-00-00 577.2 2218-273X 10.3390/biom16071013 285115907 RAZ_Nahtigal_Katarina_2026.pdf RAZ_Nahtigal_Katarina_2026.pdf 1 0907BBD3B9B468D7CAC99AF7682468A3 6c52610f0d6aab07b7b750aac7af7807a801ecf0e4bf8f4b4a996445987c46a6 7af52193-819d-11f1-9b0d-0050569b8976 https://repozitorij.uni-lj.si/Dokument.php?lang=slv&id=240298 https://www.mdpi.com/2218-273X/16/7/1013 1 05fbb73e-819d-11f1-9b0d-0050569b8976 https://repozitorij.uni-lj.si/Dokument.php?lang=slv&id=240297 Medicinska fakulteta Fakulteta za računalništvo in informatiko 0 0 0