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

IF 2.1 4区 医学 Q3 PHARMACOLOGY & PHARMACY Acta Pharmaceutica Pub Date : 2024-09-14 Print Date: 2024-09-01 DOI:10.2478/acph-2024-0029
Saranda Disha-Ibrahimi, Gorazd Drevenšek, Martina Drevenšek, Janja Marc, Irena Prodan Žitnik
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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 expres sed 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.

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将 PPIA、HRPT1 和 PGK1 基因作为奥氮平治疗大鼠牙槽骨和股骨头重塑中 RT-qPCR 正常化的适当组合。
骨重塑研究中可靠的基因表达分析需要选择适当的内部对照,即用于定量实时 PCR(RT-qPCR)归一化的稳定参考基因,这是最常用的基因表达定量测量方法。即使是最广泛使用的参考基因,在不同的实验条件下,或在不同的组织类型或治疗方案中,其表达量也会发生变化,因此选择适当的对照是确保结果可靠的关键一步。本研究旨在为奥氮平调节大鼠骨重塑的研究确定最稳定的参考基因。从奥氮平或安慰剂治疗的 Wistar 大鼠的上颌骨牙槽骨和股骨中分离出 RNA,并转录成 cDNA。通过 RT-qPCR 评估了 12 个候选参考基因的表达。使用 GeNorm、NormFinder、BestKeeper 和 delta Ct 算法以及综合排名法分析了这些基因的表达情况。PPIA、HRPT1 和 PGK1 是表达最稳定的参考基因,这三个基因的组合是归一化的最佳选择。本研究首次为奥氮平暴露大鼠的研究确定了最佳参考基因,为今后提高骨研究中 RT-qPCR 表达的准确性和可靠性提供了重要基准。
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来源期刊
Acta Pharmaceutica
Acta Pharmaceutica PHARMACOLOGY & PHARMACY-
CiteScore
5.20
自引率
3.60%
发文量
20
审稿时长
>12 weeks
期刊介绍: AP is an international, multidisciplinary journal devoted to pharmaceutical and allied sciences and contains articles predominantly on core biomedical and health subjects. The aim of AP is to increase the impact of pharmaceutical research in academia, industry and laboratories. With strong emphasis on quality and originality, AP publishes reports from the discovery of a drug up to clinical practice. Topics covered are: analytics, biochemistry, biopharmaceutics, biotechnology, cell biology, cell cultures, clinical pharmacy, drug design, drug delivery, drug disposition, drug stability, gene technology, medicine (including diagnostics and therapy), medicinal chemistry, metabolism, molecular modeling, pharmacology (clinical and animal), peptide and protein chemistry, pharmacognosy, pharmacoepidemiology, pharmacoeconomics, pharmacodynamics and pharmacokinetics, protein design, radiopharmaceuticals, and toxicology.
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