Background Endothelial nitric oxide synthase (eNOS) plays an important role in erectile physiology through nitric oxide production in penile vascular tissue. Reduced eNOS expression has been associated with diabetes-related erectile dysfunction, making reliable molecular detection of this gene important for experimental studies. This study aimed to design and validate PCR primers targeting the eNOS gene in Rattus norvegicus corpus cavernosum tissue for future gene expression applications. Methods Three primer pairs (N1, N2, and N3) were designed using NCBI Primer-BLAST based on the Rattus norvegicus eNOS reference sequence (NM₀21838. 2). Total RNA was extracted from corpus cavernosum tissue of Sprague-Dawley rats, followed by cDNA synthesis. Primer performance was evaluated using high-resolution melting (HRM) analysis across an annealing temperature range of 50–61 °C. Amplicon specificity was assessed by conventional PCR, agarose gel electrophoresis, and Sanger sequencing, followed by sequence alignment using BLAST. Results All primer pairs generated detectable amplification products, with optimal annealing observed at 57 °C. Agarose gel electrophoresis demonstrated single bands corresponding to expected product sizes of 869 bp (N1), 944 bp (N2), and 874 bp (N3). Sequence alignment confirmed target specificity, with identity values of 99. 35% for N1, 97. 77% for N2, and 100. 00% for N3 relative to the reference sequence. Among the three candidates, primer pair N3 showed the highest sequence specificity and the most consistent amplification profile. Conclusions The three primer pairs were successfully validated for conventional PCR amplification of the eNOS gene in Rattus norvegicus. Primer pair N3 demonstrated the highest specificity and may serve as a reference candidate for further molecular studies involving eNOS detection in experimental erectile dysfunction models. Additional optimization is required before application in quantitative PCR assays.
Hadibrata et al. (2026) studied this question.