Long-Term Stability of Preservative-Free Urine Samples: Superior Biomolecular Integrity at −80 °C and in Lyophilized Form

Background: Proper storage of biofluids is critical to preserving their molecular integrity for downstream applications. This study investigates the effect of different storage temperatures on the stability of preservative-free urine samples over a two-year period. Methods: Urine samples were collected, aliquoted, and stored at −80 °C, −20 °C, 4 °C, and in lyophilized form. Samples were retrieved at 0, 6, 12, and 24 months for analysis. DNA, RNA, and protein were isolated and evaluated using agarose and polyacrylamide gel electrophoresis. Nucleic acid quality was assessed using Nanodrop spectrophotometry and Bioanalyzer profiles. Results: A significant increase in pH and a concurrent decline in protein concentration were observed within the first six months at −20 °C and 4 °C. These changes plateaued after six months. Samples stored at −80 °C and in lyophilized form showed minimal variation in pH and retained higher protein stability. DNA quality, based on 260/280 and 260/230 ratios and electrophoretic band integrity, was well-preserved under these two conditions. RNA quality remained stable for up to 12 months but declined thereafter. Conclusions: Storage at −80 °C or in lyophilized form offers optimal preservation of protein concentration and nucleic acid quality in preservative-free urine samples over extended storage durations. However, lyophilization offers a cost-effective and logistically practical alternative, as samples can be stored at room temperature without the requirement of ultra-low freezers.