Cancer Diagnosis Program | Biorepositories and Biospecimen Research Branch

Search BRD Papers

Estimation of age-related DNA degradation from formalin-fixed and paraffin-embedded tissue according to the extraction methods.

Author(s): Watanabe M, Hashida S, Yamamoto H, Matsubara T, Ohtsuka T, Suzawa K, Maki Y, Soh J, Asano H, Tsukuda K, Toyooka S, Miyoshi S

Publication: Exp Ther Med, 2017, Vol. 14, Page 2683-2688

PubMed ID: 28962212 PubMed Review Paper? No

Purpose of Paper

Conclusion of Paper

Studies

1. Study Purpose

   This paper investigated the potential effects of extraction method (a silica membrane method versus total tissue DNA method) and the duration of room temperature storage of formalin-fixed, paraffin-embedded (FFPE) blocks (0.5-12 y) on DNA yield, purity, and degradation in lung adenocarcinoma specimens.  Agreement in the detection of an EGFR mutation between five case-matched FFPE and frozen specimens that were stored for up to 12 y was also explored. A total of 25 surgically resected lung adenocarcinoma tumor specimens (>10 mm diameter) were divided, and a portion was frozen at -80°C while the remainder of the specimen was fixed in 10% neutral buffered formalin (cold ischemia time not specified) overnight to 48 h at room temperature, embedded in paraffin, and stored as an FFPE block at room temperature.  Five blocks from different patients representing each storage duration timepoint (0.5, 3, 6, 9, or 12 y ) were randomly selected for the study. For each FFPE block, the area containing tumor was scraped from two 5 µm-thick slide-mounted sections into microtubes, and DNA was extracted using the silica membrane-based QIAamp DNA FFPE Tissue Kit or the WaxFree DNA Extraction Kit according to the respective manufacturer’s protocol. DNA was extracted from frozen specimens using phenol-chloroform after proteinase K digestion. DNA was quantified with a LabChip spectrophotometer (absorbance at 260 nm), the Qubit dsDNA Kit with a Qubit 2.0 fluorometer, and the Kappa QC Kit by qPCR of 41, 129, and 305 bp amplicons. DNA purity was determined by spectrophotometry (absorbance ratio of 260/280 nm) and integrity by qPCR (Q-score of amplicon sizes: Q129/Q41, the ratio of the 129 bp amplicon to the 41 bp amplicon and Q305/41, the ratio of the 305 bp amplicon to the 41 bp amplicon).  An EGFR L858R mutation was detected in DNA isolated from frozen and FFPE specimens by PCR-based sequencing of a 108 bp product.

   Summary of Findings:

   The mean DNA yield was significantly greater when FFPE lung adenocarcinoma sections were extracted using the WaxFree DNA Extraction Kit (WAX Kit) than the QIAamp DNA FFPE Tissue Kit (QIA Kit) when determined by spectrophotometry (10-fold greater, p<0.01), fluorometry (3.5-fold greater, p<0.01), and qPCR amplification of the 41 bp (but not 129 or 305 bp) fragment (2-fold greater, p<0.01). However, the purity of DNA was superior when extraction was with the QIA Kit than the WAX Kit, with significantly higher A260/280 values with the QIA Kit than the WAX Kit (2.41 versus 1.13, p<0.01).  Extraction-specific differences in DNA fragmentation were also observed, as Q129/Q41 scores (but not Q305/Q41 scores) were significantly higher after DNA extraction with the QIA Kit compared to the WAX Kit (0.27 versus 0.20, p<0.01).  The duration of FFPE block storage at room temperature did not significantly affect DNA yield (determined by spectrophotometry) or DNA quality (A260/280) when either extraction kit was used, but age-related increases in DNA fragmentation were observed for both extraction methods. FFPE blocks stored for 0.5 y had significantly higher Q-scores for Q129/Q41 and Q305/Q41 than blocks stored for 3 y when DNA was extracted using either the QIA or WAX Kit (p<0.05 for all).  FFPE blocks stored for 9 y also had significantly higher Q-scores for Q129/Q41 and Q305/Q41 than blocks stored for 12 y following DNA extraction with either the QIA or WAX Kit (p<0.01 for all).  There was 100% agreement in the detection of an EGFR L858R mutation in five pairs of FFPE specimens and case-matched frozen adenocarcinoma specimens, including one specimen pair stored for 12 y.

   Biospecimens

   • Tissue - Lung

   Preservative Types

   • Frozen
   • Formalin

   Diagnoses:

   • Neoplastic - Carcinoma

   Platform:

   Analyte	Technology Platform
   DNA	Spectrophotometry
   DNA	Fluorometry
   DNA	Real-time qPCR
   DNA	Next generation sequencing

   Pre-analytical Factors:

   Classification	Pre-analytical Factor	Value(s)
   Analyte Extraction and Purification	Analyte isolation method	QIAamp DNA FFPE Tissue Kit WaxFree DNA Extraction Kit
   Biospecimen Preservation	Type of fixation/preservation	Formalin (buffered) Frozen
   Real-time qPCR Specific	Length of gene fragment	41 bp 129 bp 305 bp
   Storage	Storage duration	0.5 y 3 y 6 y 9 y 12 y

   View more