Kosin Med J.  2020 Jun;35(1):26-37. 10.7180/kmj.2020.35.1.26.

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

• Pak MG
• Roh MS

• KMID: 2503726
• DOI: http://doi.org/10.7180/kmj.2020.35.1.26

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℃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.

Keyword

Biobank; Colorectal cancer; DNA; Ischemia; Storage

Figure

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  Fig. 1 DNA purity (260/280 ratio) in 45 biobanked fresh frozen colorectal cancer tissues. A single specimen showed out of DNA purity range with 1.76 of 260/280 ratio (★). CIT = cold ischemia time; SP = storage period; CIT-1 = less than 30 minutes; CIT-2 = 31–45 minutes; CIT-3 = 46–60 minutes; SP-1 = less than 1 year; SP-2 = 2–3 years; SP-3 = 4–5 years.

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  Fig. 2 Representative DNA integrity of nine groups confirmed by an UV transilluminator following electrophoresis with 2% agarose gel. Clear high molecular bands were observed in all biobanked specimens. C = control; 1–9 = group number.

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  Fig. 3 Representative KRAS mutation status. A colorecatal cancer in group 9 (cold ischemia time 46–60 minutes and storage period 4–5 years) showed identical KRAS codon 12 mutation in both biobanked fresh frozen tissue [A] and formalin-fixed paraffin-embedded tissue [B].

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