Effect of storage temperature on nucleic acid quality in formalin-fixed paraffin-embedded tissue samples

Abstract

Formalin-fixed paraffin-embedded (FFPE) tissue samples are widely used for genomic analyses in clinical and research settings. However, prolonged storage at room temperature may compromise nucleic acid quality, potentially affecting the reliability of molecular testing. In this study, we investigated the impact of various storage temperatures and repeated temperature changes on nucleic acid quality in FFPE samples. Four surgically resected tumor cases were analyzed. Following cold ischemia times of 29–60 min, tumor tissues were sampled upon arrival at the pathology department, divided into five equal parts, and fixed in 10 % neutral buffered formalin. The resulting FFPE blocks were stored at 18 °C, 4 °C, −20 °C, −80 °C, or − 150 °C. After storage, the blocks were returned to room temperature for sectioning and analysis. DNA and RNA quality were assessed at 3, 6, and 12 months using DNA integrity number (DIN), short-to-long cycle threshold (S/L Ct) ratio, RNA integrity number (RIN), and DV200. Samples stored at 18 °C and 4 °C exhibited time-dependent deterioration in certain indicators, whereas those stored at −20 °C or below maintained stable nucleic acid quality. Despite multiple freeze-thaw cycles, no significant degradation was observed under colder conditions. Our findings suggest that −20 °C storage is a feasible and effective approach for preserving nucleic acid integrity in FFPE tissues. Although limited by a small sample size (n = 4), this study provides practical insights into tissue archiving strategies and supports selective implementation of refrigerated storage for long-term molecular analysis.