Original scientific paper

https://doi.org/10.2478/acph-2024-0029

PPIA, HRPT1, and PGK1 genes as the appropriate combination for RT-qPCR normalization in alveolar and femoral bone remodeling in olanzapine-treated rats

SARANDA DISHA-IBRAHIMI ; Department of Orthodontics, Faculty of Medicine, University of Ljubljana, Ljubljana, Slovenia; Department of Periodontology and Oral Medicine, Faculty of Medicine, University of Prishtina, Prishtina, Kosovo
GORAZD DREVENŠEK ; Institute of Pharmacology and Experimental Toxicology, Faculty of Medicine, University of Ljubljana, Ljubljana, Slovenia; Department of Psychology, Faculty of Mathematics, Natural Sciences and Information Technologies, University of Primorska, Koper, Slovenia
MARTINA DREVENŠEK ; Department of Orthodontics, Faculty of Medicine, University of Ljubljana, Ljubljana, Slovenia; Department of Orthodontics, University Medical Centre Ljubljana, Ljubljana, Slovenia *
JANA MARC ; Department of Clinical Biochemistry, Faculty of Pharmacy, University of Ljubljana, Ljubljana, Slovenia
IRENA PRODAN ŽITNIK ; Department of Clinical Biochemistry, Faculty of Pharmacy, University of Ljubljana, Ljubljana, Slovenia

* Corresponding author.

Abstract

Reliable gene expression analysis in bone remodeling studies requires an appropriate selection of internal controls, i.e. stable reference genes for the normalization of quantitative real-time PCR (RT-qPCR), the most common method used for quantifying gene expression measurements. Even the most widely used reference genes can have variable expression under different experimental conditions, or in different tissue types or treatment regimes, so selecting appropriate controls is a key step in ensuring reliable results. The aim of this research was to identify the most stable reference gene(s) for the study of olanzapine modulated bone remodeling in rats. RNA was isolated from the maxillary alveolar and femoral bones of olanzapine or placebo-treated Wistar rats and transcribed to cDNA. The expression of 12 candidate reference genes was assessed by RT‐qPCR. Their expressions were analysed using GeNorm, NormFinder, BestKeeper and delta Ct algorithms, and by the comprehensive ranking method. PPIA, HRPT1 and PGK1 were the most stably expressed reference genes and the combination of the three genes was optimal for normalization. This study is the first to identify the optimal reference genes for research in olanzapine-exposed rats, which serve as a pivotal benchmark for enhancing the accuracy and reliability of future RT-qPCR expression in bone studies.

Keywords

reference genes; alveolar bone; RT-qPCR; rat; olanzapine

Hrčak ID:

320043

URI

https://hrcak.srce.hr/320043

Publication date:

30.9.2024.

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