Reference genes for circadian profiling of core clock genes in the blood of obstructive sleep apnea patients

Nahtigal, Katarina (author), Halužan Vasle, Ana (author), Kreft, Tinkara (author), Skubic, Cene (author), Mraz, Miha (author), Moškon, Miha (author), Dolenc-Grošelj, Leja (author), Rozman, Damjana (author)

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.

• Language: English
• Keywords: circadian rhythms, obstructive sleep apnea, reference genes, qRT-PCR normalization, gene expression stability, core clock genes
• Work type: Article
• Typology: 1.01 - Original Scientific Article
• Organization: MF - Faculty of Medicine; FRI - Faculty of Computer and Information Science
• Publication status: Published
• Publication version: Version of Record
• Year: 2026
• Number of pages: 18 str.
• Numbering: Vol. 16, iss. 7, art. 1013
• PID: 20.500.12556/RUL-184903
• UDC: 577.2
• ISSN on article: 2218-273X
• DOI: 10.3390/biom16071013
• COBISS.SI-ID: 285115907
• Publication date in RUL: 17.07.2026

Record is a part of a journal

• Title: Biomolecules
• Publisher: MDPI
• ISSN: 2218-273X
• COBISS.SI-ID: 519952921

Licences

• License: CC BY 4.0, Creative Commons Attribution 4.0 International
• Link: http://creativecommons.org/licenses/by/4.0/
• Description: This is the standard Creative Commons license that gives others maximum freedom to do what they want with the work as long as they credit the author.

Secondary language

• Language: Slovenian
• Keywords: cirkadiani ritmi, obstruktivna apneja v spanju, referenčni geni, normalizacija qRT-PCR, stabilnost izražanja genov, osrednji geni uravnavanja ritma, ključni geni biološke ure