Urinary miRNA Analysis for Clear Cell Renal Cell Carcinoma: miR-20a as a Key Endogenous Normalizer

Urinary microRNAs (miRNAs) are promising noninvasive biomarkers for cancer detection, but their clinical utility is reduced by inconsistent normalization strategies, reducing reproducibility and comparability across studies. In this study, we assessed the stability of miR-20a as an endogenous normalizer for urinary miRNA profiling in clear cell renal cell carcinoma (ccRCC) while standardizing the pre-analytical phase using a urine stabilizing solution. Ninety-nine urine samples were analyzed: 47 from healthy individuals, 30 from ccRCC patients pre-surgery, and 22 post-operative patients. Six candidate miRNAs-miR-20a, miR-15b, miR-16, miR-15a, miR-210-3p, and miR-let-7b-were quantified via RT-qPCR. Stability analysis with RefFinder, integrating multiple algorithms (geNorm, normFinder, BestKeeper, and ΔCt methods), identified miR-20a as the most stable among the six candidates. Raw Ct values of miR-20a were normally distributed (Shapiro-Wilk test, p > 0.05), with no significant intergroup differences (one-way ANOVA, F(2.96) = 2.324, p = 0.103) and minimal intragroup variability (CV% 4.98-6.38). MiR-20a expression remained stable across different tumor staging, grading, and urine storage durations. These findings confirm miR-20a as a robust endogenous normalizer for urinary miRNA analyses and support the feasibility of developing reproducible urinary liquid biopsy workflows for ccRCC, even in settings where immediate sample processing is not feasible.