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

Autor: Gnanaskandan S Sr; Microbiology, Sri Ramachandra Institute of Higher Education and Research, Chennai, IND., Karunakaran U; Virology, Manipal Institute of Virology, Manipal, IND., Srikanth P; Microbiology, Retired-Private Practice, Chennai, IND.
Jazyk: angličtina
Zdroj: Cureus [Cureus] 2024 Nov 29; Vol. 16 (11), pp. e74715. Date of Electronic Publication: 2024 Nov 29 (Print Publication: 2024).
DOI: 10.7759/cureus.74715
Abstrakt: 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 × 10 10 copies/ml, followed by serial dilutions to achieve a range from 10 6 to 10 2 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.
Competing Interests: Human subjects: Consent for treatment and open access publication was obtained or waived by all participants in this study. Institutional Ethics Committee, Sri Ramachandra Institute of Higher Education and Research, Porur, Chennai issued approval IEC-NI/18/JAN/63/06. Animal subjects: All authors have confirmed that this study did not involve animal subjects or tissue. Conflicts of interest: In compliance with the ICMJE uniform disclosure form, all authors declare the following: Payment/services info: All authors have declared that no financial support was received from any organization for the submitted work. Financial relationships: All authors have declared that they have no financial relationships at present or within the previous three years with any organizations that might have an interest in the submitted work. Intellectual property info: A patent has been filed for our designed primers and probe titled "A Novel Assay for Quantification of Nf-Kappa B P65 Expression to Determine Physiological and Pathological Conditions" under Intellectual Property India. The allotted patent application number from the patent office is 202441054401 with a filing date of 16/07/2024. Other relationships: All authors have declared that there are no other relationships or activities that could appear to have influenced the submitted work.
(Copyright © 2024, Gnanaskandan et al.)
Databáze: MEDLINE
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