Chen Siyu, Cai Qi, Liu Peipei, Liu Jingru, Chen Guanzhou, Yan Hanwei, Zheng Han
National Engineering Laboratory of Crop Stress Resistance Breeding, School of Life Sciences, Anhui Agricultural University, Hefei 230026, China.
Laboratory of Modern Biotechnology, School of Forestry and Landscape Architecture, Anhui Agricultural University, Hefei 230026, China.
Curr Issues Mol Biol. 2024 Dec 18;46(12):14304-14320. doi: 10.3390/cimb46120857.
Quantitative real-time PCR (qRT-PCR) is an essential tool for analyzing and selecting stable reference genes. In order to screen for suitable reference genes under high-temperature stress conditions in , this study measured the relative expression levels of 17 candidate reference genes using qRT-PCR. Among these, four are traditional reference genes, while the remaining thirteen are candidate reference genes with no previous reports on their use as reference genes. The expression stability of the candidate reference gene expression was analyzed and evaluated using five methods: ΔCt, geNorm, NormFinder, BestKeeper, and RefFinder. The results indicated that the and genes displayed the highest stability in expression under high-temperature stress conditions. To verify the stability of the reference genes, we treated with high-temperature stress, used the selected and as references, and analyzed the expression of the heat-responsive gene using qRT-PCR. The results showed that when and were used individually or in combination as reference genes, the relative expression of significantly increased and remained consistent under high-temperature treatment. This indicates that both and are suitable reference genes for qRT-PCR analysis in exposed to high-temperature stress. The novel reference genes identified in this study will serve as a reliable reference standard for gene expression studies in under high-temperature stress, thereby enhancing the accuracy and comparability of experimental data.
定量实时聚合酶链反应（qRT-PCR）是分析和选择稳定参考基因的重要工具。为了筛选出在[具体研究对象]高温胁迫条件下合适的参考基因，本研究使用qRT-PCR测量了17个候选参考基因的相对表达水平。其中，4个是传统参考基因，其余13个是此前尚无作为参考基因使用报道的候选参考基因。使用ΔCt、geNorm、NormFinder、BestKeeper和RefFinder这五种方法对候选参考基因表达的稳定性进行了分析和评估。结果表明，[具体基因1]和[具体基因2]基因在高温胁迫条件下表达稳定性最高。为验证参考基因的稳定性，我们对[具体研究对象]进行高温胁迫处理，选用筛选出的[具体基因1]和[具体基因2]作为参考基因，并使用qRT-PCR分析热响应基因[具体基因3]的表达。结果显示，当单独或联合使用[具体基因1]和[具体基因2]作为参考基因时，[具体基因3]的相对表达在高温处理下显著增加且保持一致。这表明[具体基因1]和[具体基因2]都是[具体研究对象]在高温胁迫下进行qRT-PCR分析的合适参考基因。本研究中鉴定出的新参考基因将作为[具体研究对象]在高温胁迫下基因表达研究的可靠参考标准，从而提高实验数据的准确性和可比性。
