Improved results of LINE-1 methylation analysis in formalin-fixed, paraffin-embedded tissues with the application of a heating step during the DNA extraction process.
Author(s): Wen X, Jeong S, Kim Y, Bae JM, Cho NY, Kim JH, Kang GH
Publication: Clin Epigenetics, 2017, Vol. 9, Page 1
PubMed ID: 28149329 PubMed Review Paper? No
Purpose of Paper
This paper investigated potential effects of heat-mediated denaturation on DNA methylation using formalin-fixed paraffin-embedded (FFPE) lymph node and gastric tumor specimens.
Conclusion of Paper
Heating DNA samples prior to bisulfate conversion decreased methylation levels of 3 of 4 of the LINE-1 CPG sites, but also reduced the standard deviation of LINE-1 methylation levels and increased the level and frequency of methylation for 7 of the eight individual CpG islands in the methylator phenotype panel (CIMP). Thus, the authors conclude that including a heating step prior to bisulfate conversion minimizes FFPE-induced artifacts that affect quantification of LINE-1 methylation.
Studies
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Study Purpose
This study explored how DNA methylation is affected by heat-mediated denaturation using DNA extracted from 476 archival FFPE lymph node specimens removed from patients diagnosed with advanced gastric cancer that were stored for 6 to 7 years and 497 FFPE colorectal cancer specimens that were stored for 4 to 10 years. FFPE specimens were deparaffinized with xylene, microdissected to obtain an area of high tumor density, and Proteinase K digested for 2 days at 55˚C. Lysates were then heat-treated at 95˚C for 30 min, while controls were held at room temperature, and subjected to bisulfite conversion with the EZ DNA methylation kit. Methylation was then determined by pyrosequencing of the converted DNA.
Summary of Findings:
Heating DNA isolated from FFPE lymph node to 95˚C prior to bisulfate conversion decreased overall methylation levels for three of the four LINE-1 CpG sites evaluated but also reduced the standard deviation of all four LINE-1 CpG sites. In colorectal carcinoma specimens, heating DNA lysates to 95˚C increased both the level and frequency of methylation for 7 of eight individual CpG islands represented on the methylator phenotype panel (CIMP), thereby improving detection of CIMP-high colorectal cancer. The lowest levels of LINE-1 methylation observed were associated with decreases in overall survival and disease-free survival, regardless of whether DNA was heated although samples that were heated displayed lower P-values than those that were not. While freshly frozen and FFPE xenografts differed in overall methylation levels, the increase in methlyation observed for FFPE specimens was eliminated by heat treatment for immediately fixed FFPE specimens but not those subjected to a 1-2 d room temperature delay to fixation.
Biospecimens
Preservative Types
- Formalin
Diagnoses:
- Neoplastic - Carcinoma
Platform:
Analyte Technology Platform DNA Bisulfite conversion assay Pre-analytical Factors:
Classification Pre-analytical Factor Value(s) Bisulfite conversion assay Specific Template modification Heated to 95˚C for 30 min
Not heated