Cancer Diagnosis Program | Biorepositories and Biospecimen Research Branch

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

Author(s): Pak MG, Roh MS

Publication: Kosin Medical Journal, 2020, Vol. 35, Page 26-37

Review Paper? No

Purpose of Paper

Conclusion of Paper

Studies

1. Study Purpose

   The purpose of this paper was to assess the potential effects associated with a cold ischemia time of up to 1 h at room temperature and the duration of frozen storage on the quality and amplificability of DNA isolated from colorectal tumor specimens. In total, surgically resected colorectal tumor specimens from 45 patients with colorectal cancer were sectioned into 0.5 cm³ pieces and subjected to a cold ischemia time at room temperature of <30 min, 30-45 min, or 45-60 min, frozen at -80°C in cryovials; five specimens from each cold ischemia timepoint were then stored at -80°C for the following durations:  <1 y, 2-3 y, 4-5 y.  Prior to DNA extraction, frozen CRC tumor specimens were embedded in OCT, sectioned at a thickness of 5 µm, and then washed with PBS to remove the OCT compound. Sections were incubated in cell lysis buffer, treated with proteinase K (concentration and duration not specified), and DNA was extracted using the paramagnetic bead-based Maxwell 16 Viral Total Nucleic Acid Purification Kit.  DNA purity was determined by NanoDrop spectrophotometry, and absorbance ratio (260/280 nm) thresholds were set at >1.8 (<1.8 was indicative of protein or phenol contamination) and <2.0 (> 2.0 was indicative of RNA contamination). DNA integrity was evaluated by gel electrophoresis.  KRAS mutation (exon 2, codon 12 and 13) status was assayed by real-time PCR (CFX96 PCR detection system) with the PNAClamp KRAS Mutation Detection Kit. Cycle threshold (Ct) values were used to identify the presence of a KRAS mutation (ΔCt threshold = 2.0).

   Summary of Findings:

   DNA purity was not affected by a cold ischemia time at room temperature of up to 1 h (1.80-1.90) or frozen storage of CRC tumor specimens at -80°C for up to 5 y (1.87-1.88); only one specimen (cold ischemia time, 31-45 min; frozen storage duration, 2-3 y) had a 260/280 ratio that fell outside of the thresholds specified (1.76). DNA from all CRC tumor specimens were considered of good quality; a crisp high molecular weight band was observed for all cold ischemia times and storage durations and low molecular weight smears were not observed. Frozen CRC tumor specimens that experienced up to 1 h of cold ischemia time and were stored at -80°C for up to 5 y displayed 100% concordance in KRAS mutation status with case-matched FFPE tumor specimens obtained and analyzed at the time of diagnosis.

   Biospecimens

   • Tissue - Colorectal

   Preservative Types

   • Frozen
   • OCT

   Diagnoses:

   • Neoplastic - Carcinoma

   Platform:

   Analyte	Technology Platform
   DNA	Spectrophotometry
   DNA	Electrophoresis
   DNA	Real-time qPCR

   Pre-analytical Factors:

   Classification	Pre-analytical Factor	Value(s)
   Biospecimen Acquisition	Cold ischemia time	<30 min 30-45 min 45-60 min
   Storage	Storage duration	<1 y 2-3 y 4-5 y
   Real-time qPCR Specific	Targeted nucleic acid	KRAS
   Biospecimen Preservation	Type of fixation/preservation	Formalin (buffered) Frozen

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