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Updated: Jun 15, 2026
Measuring Dengue Virus RNA in the Culture Supernatant of Infected Cells by Real-time Quantitative Polymerase Chain Reaction
Published on: November 1, 2018
Endogenous gene selection for relative quantification PCR and IL6 transcript levels in the PBMC's of severe and
Vigneshwari Easwar Kumar1, Cleetus Cherupanakkal2, Minna Catherine1
1Central Inter-Disciplinary Research Facility, Sri Balaji Vidyapeeth (Deemed to be University), Puducherry, 607402, India.
Objectives:
Dengue viral infection ranges from dengue fever to dengue haemorrhagic fever and lethal dengue shock syndrome. Currently no means are available to monitor the progression of disease. Real time PCR based gene expression analyses are used to find potential molecular markers for effective prediction of dengue clinical outcome. The accuracy of qPCR analysis is strongly dependent on transcript normalization using stably expressed endogenous genes, which if selected imprecisely can lead to misinterpreted results. We aimed to determine the best fit for endogenous gene among six genes namely COX, ACTB, GAPDH, HMBS, HPRT and B2M for dengue viral infection cases. Gene stability was inferred from qPCR data by normalizing with two algorithms geNorm and Normfinder and the rankings generated were validated by gene expression analysis against target gene IL-6.
Results:
Both the algorithms showed ACTB, HPRT, GAPDH as most stable genes. Normalizing with the stable genes revealed a significant fold change (p < .05) in IL-6 levels of .32, .52, .69, and .62 in non-dengue febrile illness, non severe, severe and All Dengue groups respectively compared to healthy controls. based on our study, we suggest ACTB with HPRT/GAPDH combination for normalization in qPCR for precise quantification of transcripts in dengue infected studies.
