Quantitative Evaluation of Endogenous Reference Genes for RT-qPCR and ddPCR Gene Expression Under Polyextreme Conditions Using Anaerobic Halophilic Alkalithermophile Natranaerobius thermophilus
文献类型： 外文期刊
作者： Tao, Xinyi 1 ; Xing, Qinghua 1 ; Zhang, Yingjie 1 ; Atnkut, Belsti 2 ; Wei, Haozhuo 1 ; Ramirez, Silva 1 ; Mao, Xinwei 3 ; Zhao, Baisuo 1 ;
作者机构： 1.Chinese Acad Agr Sci, Grad Sch, Beijing 100081, Peoples R China
2.Injibara Univ, Coll Nat & Computat Sci, Dept Biol, POB 40, Injibara, Ethiopia
3.SUNY Stony Brook, Dept Civil Engn, Stony Brook, NY 11794 USA
关键词：
endogenous reference genes; RT-qPCR; polyextreme condition; extremophiles;
期刊名称：MICROORGANISMS （ 影响因子：4.2； 五年影响因子：4.6 ）
ISSN：
年卷期： 2025 年 13 卷 8 期
页码：
收录情况： SCI
摘要： Accurate gene expression quantification using reverse transcription quantitative PCR (RT-qPCR) requires stable reference genes (RGs) for reliable normalization. However, few studies have systematically identified RGs suitable for simultaneous high salt, alkaline, and high-temperature conditions. This study addresses this gap by evaluating the stability of eight candidate RGs in the anaerobic halophilic alkalithermophile Natranaerobius thermophilus JW/NM-WN-LFT under combined salt, alkali, and thermal stresses. The stability of these candidate RGs was assessed using five statistical algorithms: Delta CT, geNorm, NormFinder, BestKeeper, and RefFinder. Results indicated that recA exhibited the highest expression stability across all tested conditions and proved adequate as a single RG for normalization in both RT-qPCR and droplet digital PCR (ddPCR) assays. Furthermore, recA alone or combined with other RGs (sigA, rsmH) effectively normalized the expression of seven stress-response genes (proX, opuAC, mnhE, nhaC, trkH, ducA, and pimT). This work represents the first systematic validation of RGs under polyextreme stress conditions, providing essential guidelines for future gene expression studies in extreme environments and aiding research on microbial adaptation mechanisms in halophilic, alkaliphilic, and thermophilic microorganisms.
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