Kruszewska-Naczk Beata, Grinholc Mariusz, Rapacka-Zdonczyk Aleksandra
Laboratory of Photobiology and Molecular Diagnostics, Intercollegiate Faculty of Biotechnology, University of Gdansk and Medical University of Gdansk, Gdańsk, Poland.
Front Mol Biosci. 2025 Jan 6;11:1467726. doi: 10.3389/fmolb.2024.1467726. eCollection 2024.
Reverse transcription quantitative real-time polymerase chain reaction Q7 (RT‒qPCR) is a commonly used tool for gene expression quantification. Because the qPCR method depends on several variables that can influence the analysis process, stably expressed genes should be selected for relative gene expression studies. To date, there is insufficient information on the selection of appropriate reference genes for antimicrobial photodynamic inactivation (aPDI) and antimicrobial blue light (aBL) treatment. Therefore, the purpose of the present study was to determine the most stable reference gene under treatment with aBL under sublethal conditions and to evaluate differences in the expression of the selected gene after aBL treatment in comparison to the nontreated control.
Selection of stable reference genes was performed using 4 programs: BestKeeper, geNorm, NormFinder and RefFinder under 409 and 415 nm aBL treatment.
The results revealed that the gene encoding the integration host factor β subunit (ihfB) in was the most stably expressed gene after both 409 and 415 nm aBL treatment. Three programs, RefFinder, geNorm, and NormFinder, indicated that this gene had the most stable expression in comparison to the other reference gene candidates. The next best candidates were cysG, uidA, and gyrA. NormFinder revealed ihfB as the single gene and cysG - gyrA as the combination of reference genes with the best stability.
Universal reference genes are characterized by stable expression that remains consistent across various stress conditions. Consequently, it is essential to evaluate reference genes for each specific stress factor under investigation. In the case of aBL at different wavelengths, we identified genes that maintain stable expression following irradiation.
逆转录定量实时聚合酶链反应（RT-qPCR）是一种常用的基因表达定量工具。由于qPCR方法依赖于几个可能影响分析过程的变量，因此在进行相对基因表达研究时应选择稳定表达的基因。迄今为止，关于抗微生物光动力灭活（aPDI）和抗微生物蓝光（aBL）治疗中合适内参基因选择的信息不足。因此，本研究的目的是确定亚致死条件下aBL处理后最稳定的内参基因，并评估aBL处理后所选基因与未处理对照相比表达的差异。
在409和415nm aBL处理下，使用BestKeeper、geNorm、NormFinder和RefFinder这4个程序选择稳定的内参基因。
结果显示，在409和415nm aBL处理后，编码整合宿主因子β亚基（ihfB）的基因是最稳定表达的基因。RefFinder、geNorm和NormFinder这3个程序表明，与其他候选内参基因相比，该基因具有最稳定的表达。其次是cysG、uidA和gyrA。NormFinder显示ihfB为单一基因，cysG-gyrA为稳定性最佳的内参基因组合。
通用内参基因的特点是在各种应激条件下表达稳定且保持一致。因此，有必要针对所研究的每个特定应激因素评估内参基因。对于不同波长的aBL，我们鉴定出了照射后保持稳定表达的基因。
