Preanalytical Determinants of DNA and RNA Quality in FFPE Tissues: Practical Recommendations for Molecular Testing

FFPE tissue is essential for molecular testing, but nucleic acid quality is affected by preanalytical factors. Using porcine models, we systematically examined how formalin concentration, fixation duration, cold ischemia time, and tissue size affect DNA and RNA quality. Fresh liver was trimmed to 2, 10, or 50 mm and fixed in 10% or 20% neutral-buffered formalin (NBF) for 1, 3, or 7 days. In small samples, 10% NBF preserved DNA quality better than 20% NBF, while this difference was absent in large samples. RNA quality showed minimal susceptibility to formalin concentration. Both DNA and RNA deteriorated as fixation duration increased from 1 to 3 to 7 days. We next evaluated the impact of post-section storage using clinical FFPE specimens. DNA and RNA fragmentation increased after > 1 month and > 3 months, respectively, which was prevented by storage at ≤ 4°C. FFPE blocks < 1 year old yielded optimal DNA quality. These findings provide practical recommendations: (1) use 10% NBF with ≤ 24-h fixation; 20% NBF may be acceptable for large specimens, although its applicability to diverse specimen types remains unverified, (2) select blocks < 1 year old, (3) section within 1 month before testing, (4) store sections at ≤ 4°C when storage exceeds 1 month.