Agrawal Nishtha, Khanna Madhu, Dhawan Gagan
Department of Virology (a Unit of Dept. of Microbiology), Vallabhbhai Patel Chest Institute, University of Delhi, Delhi, 110007, India; Department of Biomedical Science, Acharya Narendra Dev College, University of Delhi, Kalkaji, New Delhi, 110019, India.
Department of Virology (a Unit of Dept. of Microbiology), Vallabhbhai Patel Chest Institute, University of Delhi, Delhi, 110007, India.
Indian J Med Microbiol. 2023 Mar-Apr;42:49-52. doi: 10.1016/j.ijmmb.2023.01.007. Epub 2023 Feb 3.
Quantitative PCR (qPCR) is a reliable and robust technique for gene expression analysis, but its efficacy is dependent on the normalization of qPCR data with the stably expressed reference gene. Selection of a suitable reference gene is mandatory for accurate gene expression analysis, till data the most appropriate reference gene during chikungunya virus infection has not been elucidated.
In this study the expression of reference genes(GAPDH, GUSB, HPRT, Beta-actin, 18S rRNA) was analysed during chikungunya virus infection by quantitative PCR. The stability of the house-keeping genes was evaluated with three bioinformatics softwares: BestKeeper, NormFinder and GeNorm.
The significant variation in the expression of house-keeping genes (GusB, Beta-actin, HPRT) was observed during chikungunya virus infection; whereas GAPDH and 18S rRNA was most stable. The stability of reference genes analysed by the bioinformatics software further corroborate the results of qPCR.
This is first study that identifies and validates the most suitable reference gene for normalization of qPCR data during chikungunya based gene expression analysis. This could serve as a reference study for the researchers working on different aspects of chikungunya virus infections.
定量聚合酶链反应（qPCR）是一种用于基因表达分析的可靠且强大的技术，但其有效性取决于使用稳定表达的参考基因对qPCR数据进行标准化。选择合适的参考基因对于准确的基因表达分析至关重要，然而，在基孔肯雅病毒感染期间最合适的参考基因尚未明确。
在本研究中，通过定量PCR分析了基孔肯雅病毒感染期间参考基因（甘油醛-3-磷酸脱氢酶（GAPDH）、β-葡萄糖醛酸酶（GUSB）、次黄嘌呤磷酸核糖转移酶（HPRT）、β-肌动蛋白、18S核糖体RNA）的表达。使用三种生物信息学软件（BestKeeper、NormFinder和GeNorm）评估管家基因的稳定性。
在基孔肯雅病毒感染期间观察到管家基因（GusB、β-肌动蛋白、HPRT）表达存在显著差异；而GAPDH和18S rRNA最为稳定。生物信息学软件分析的参考基因稳定性进一步证实了qPCR的结果。
这是第一项在基于基孔肯雅病毒的基因表达分析中鉴定并验证用于qPCR数据标准化的最合适参考基因的研究。这可为从事基孔肯雅病毒感染不同方面研究的人员提供参考。
