Influence of Cold Ischemia Time and Storage Period on DNA Quality and Biomarker Research in Biobanked Colorectal Cancer Tissues

Abstract

Objectives Biobanking plays an important role in future research. Assessment and control of the preanalytical variables of biobanked tissues are fundamentals for the optimal use of biospecimens. Methods Forty-five colorectal cancer (CRC) tissues stored at −80°C in Bio-Resource Bank were evaluated to define the influence of cold ischemia time (CIT) and storage period (SP) on DNA quality in biobanked tissues. Three CITs (less than 30 minutes (CIT-1), 30–45 minutes (CIT-2), and 45–60 minutes (CIT-3)) and three SPs (less than 1 year (SP-1), 2–3 years (SP-2), and 4–5 years (SP-3)) were chosen. NanoDrop spectrophotometer was used to determine the 260/280 ratio for DNA purity. DNA integrity was analyzed by a UV transilluminator following electrophoresis on 2% agarose gel. To evaluate the practical usability of DNA for biomarker research, KRAS mutation status was assessed by PCR amplification. Results All DNA specimens had a 260/280 ratio ranging between 1.8 and 2.0 with the exception of one specimen (CIT-2/SP-2 group). For DNA integrity, DNA appeared as a compact, high-molecular-weight band with no or scanty low-molecular-weight smears. The concordance of KRAS mutation status between paired biobanked frozen tissues and formalin-fixed paraffin-embedded tissues was 100%. DNA remained stable in CRC tissues kept at room temperature for up to 1 hour and long-term storage up to 5 years. Conclusions Storage conditions of our biobank are suitable for long-term (at least five years) specimen preservation with high DNA quality. These results have practical implications that could affect banking guidelines.

Keywords

• biobank
• colorectal cancer
• dna
• ischemia
• storage