Design and In silico Validation of Primers for Assessing Gene Expression in the Canonical Inflammatory Pathway

Abstract

Aims: This study aimed to design and validate primers to investigate the expression of key genes of the canonical inflammation pathway, specifically NEMO (NF-kappaB Essential Modulator), MyD88 and IL-1β, in order to ensure feasibility, specificity and efficiency for use in RT-qPCR. Study Design:  This is an experimental study aimed at the design and validation of primers using in silico strategies. Methodology: FASTA sequences for key genes of the canonical inflammation pathway were obtained from the NCBI database and open reading frames (ORFs) were identified using the ORF Finder tool, while conserved domains were annotated using the NCBI conserved domains database. Primers were designed using OligoPerfect Primer Designer (ThermoFisher) based on parameters such as primer length (18–22 bp), GC content (40–60%) and melting temperature (57–63 °C). The primers were further validated in silico using OligoAnalyzer (IDTDNA) to assess their thermodynamic properties, such as hairpin formation and self-dimers, as well as confirmation of specificity by NCBI BLAST analysis. Results: The designed primers exhibited optimal characteristics for gene amplification by PCR, including hairpin formation temperatures between 35.4°C and 49.5°C and melting temperatures above 63°C. The amplicons ranged from 88 to 136 bp, which is considered an acceptable standard for qPCR analysis. Pairwise analysis on the NCBI BLAST platform confirmed species specificity for Mus musculus, with the genes of interest showing additional compatibility with Rattus norvegicus in two (Myd88 and IL 1β) of the three primers. These findings support the applicability of the primers to detect and quantify transcripts of key genes of the canonical inflammatory pathway in tissue from laboratory animal specimens. Conclusion: The validated primers provide the possibility for the amplification and quantification of key genes in the canonical inflammation pathway. This methodology ensures sensitivity and specificity for gene expression studies in the Mus Muscullus animal model, supporting future experimental applications in preclinical models of inflammation and  autoimmunity.