Acharya Arpit, Tripathi Gayatri, Rasal Kiran D, Pathan Mujahidkhan A, Bhat Raja Aadil Hussain, Dinakaran Chandana, Rathinam R Bharathi
Indian Council of Agricultural Research, Central Institute of Fisheries Education, Mumbai, Maharashtra, India.
Indian Council of Agricultural Research, Directorate of Coldwater Fisheries and Research, Bhimtal, Uttarakhand, India.
J Aquat Anim Health. 2024 Dec;36(4):330-341. doi: 10.1002/aah.10234. Epub 2024 Dec 1.
The choice of a stable reference gene plays a crucial role in gene expression analysis. In most cases, the choice of housekeeping gene (HKG) is quite random. However, the expression of HKGs varies under different experimental conditions and in different tissues.
The study focused on three reference genes in Catla Labeo catla-beta actin (β-actin), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), and elongation factor-1 alpha (EF-1α)-under four experimental conditions: bacterial challenge, hypoxia, a combination of bacterial challenge and hypoxia, and control. The fish were subjected to the above-mentioned experimental circumstances, and five tissues (blood, gill, liver, kidney, and spleen) were collected at regular intervals.
The investigation of gene expression in the five tissues was performed using real-time (RT) quantitative polymerase chain reaction (qPCR), and the resultant cycle threshold values were subjected to several statistical techniques via the RefFinder tool to determine the most stable HKG. The results were ordered in accordance with the stability of the reference genes. In this study, GAPDH was found to be the least stable gene, while β-actin and EF-1α remained stable in the kidney and liver under various circumstances. Other tissues demonstrated tissue- and treatment-specific stability for the various HKGs.
The findings indicate that GAPDH should be avoided in Catla for gene expression studies and that different algorithms should be employed to validate the stability of HKGs in RT-qPCR analysis.
选择稳定的内参基因在基因表达分析中起着至关重要的作用。在大多数情况下，管家基因（HKG）的选择相当随意。然而，HKG的表达在不同实验条件和不同组织中会有所变化。
本研究聚焦于三种内参基因，即印度鲶鱼的β-肌动蛋白（β-actin）、甘油醛-3-磷酸脱氢酶（GAPDH）和延伸因子-1α（EF-1α），研究其在四种实验条件下的情况：细菌攻击、缺氧、细菌攻击与缺氧联合作用以及对照。将鱼置于上述实验环境中，并定期收集五个组织（血液、鳃、肝脏、肾脏和脾脏）。
使用实时（RT）定量聚合酶链反应（qPCR）对五个组织中的基因表达进行研究，并通过RefFinder工具对所得的循环阈值进行多种统计分析，以确定最稳定的HKG。结果根据内参基因的稳定性进行排序。在本研究中，发现GAPDH是最不稳定的基因，而β-actin和EF-1α在各种情况下在肾脏和肝脏中保持稳定。其他组织对各种HKG表现出组织和处理特异性的稳定性。
研究结果表明，在印度鲶鱼的基因表达研究中应避免使用GAPDH，并且在RT-qPCR分析中应采用不同算法来验证HKG的稳定性。
