The Standardization and Quantification of Nuclear Factor Kappa B p65 by Real-Time Quantitative Polymerase Chain Reaction

The accurate quantification of nuclear factor Kappa B p65 (NF-κB p65) is critical for understanding inflammatory mechanisms, especially in HIV-1 infected individuals, where NF-κB p65 contributes to chronic immune activation. Conventional methods such as enzyme-linked immunosorbent assay (ELISA) and western blotting are limited in terms of sensitivity and reproducibility. This study aimed to devise a standardized real-time quantitative polymerase chain reaction (RT-qPCR) assay for NF-κB p65 using specifically designed primers and a probe. The human NF-κB p65 sequence was obtained from the National Center for Biotechnology Information (NCBI) database, and specific primers and probes were designed using Primer3 v4.1.0 software, optimizing melting temperature and guanine-cytosine (GC) content to ensure stability. Standards were synthesized using the ImmuGenix cloning kit (ImmuGenix Biosciences Pvt. Ltd., Chennai, India) at an initial concentration of 2.8 × 10¹⁰ copies/ml, followed by serial dilutions to achieve a range from 10⁶ to 10² copies/ml. RT-qPCR for NF-κB p65 was performed using the TAKARA master mix (Takara Bio, Kusatsu, Japan). The assay precision was evaluated through intra- and inter-assay measurements, with coefficient of variation (CV%) thresholds set at <5% and <10%, respectively. NF-κB p65 levels were quantified and analyzed in HIV-1 infected individuals and uninfected healthy controls. The RT-qPCR assay showed high intra-assay precision, with CV% values ranging from 0.07% to 0.2%, indicating minimal variability within individual runs. Inter-assay reproducibility showed CV% values between 0.6% and 3.6%, confirming consistent performance across experimental runs. The cycle threshold (Ct) values for NF-κB p65 were lower among HIV-1-infected individuals, indicating higher expression compared to uninfected healthy controls. Based on our findings, our standardized RT-qPCR protocol provides a reliable and reproducible approach for quantifying NF-κB p65, aiding in the understanding of inflammatory responses.