The influence of DNA degradation in formalin-fixed, paraffin-embedded (FFPE) tissue on locus-specific methylation assessment by MS-HRM.
Author(s): Daugaard I, Kjeldsen TE, Hager H, Hansen LL, Wojdacz TK
Publication: Exp Mol Pathol, 2015, Vol. 99, Page 632-40
PubMed ID: 26551081 PubMed Review Paper? No
Purpose of Paper
This paper investigated if DNA integrity was associated with DNA yield and success of real-time PCR and methylation-sensitive high-resolution melting (MS-HRM) in formalin-fixed paraffin-embedded (FFPE) non- small cell lung cancer (NSCLC) specimens.
Conclusion of Paper
Although all 107 specimens displayed some degree of DNA degradation, those that were the most degraded generally had lower yields than those with more intact DNA. Amplification of all three promoters occurred in specimens of all quality, but the amplification curves shifted to the right with increasing degradation of the initial DNA despite starting with the same amount of bisulfite-modified DNA, indicating less amplifiable DNA in those samples. MS-HRM analysis was able to assess the methylation status of BRCA2 and miR-34a promoters in all 15 specimens examined and of the APC promoter in 14 of the 15 specimens, with the exception being a specimen with highly degraded DNA.
Studies
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Study Purpose
This study investigated the association between DNA integrity and DNA yield, and success of real-time PCR and methylation sensitive high resolution melting (MS-HRM) in FFPE NSCLC specimens. DNA was extracted from five 10 µm-thick sections of archival FFPE surgical resection NSCLC specimens (n=107) using the High Pure FFPET DNA Isolation Kit. Details of specimen processing (fixation, embedding, and storage) were not provided. Control DNA was obtained from the peripheral blood of five healthy patients using a salting-out based method. DNA concentration was determined by spectrophotometer and integrity was assessed by electrophoresis. DNA was bisulfite-converted using the EZ DNA Methylation-Gold Kit and methylation of the BRCA2, miR-34a, and APC promoters was assessed by real-time PCR amplification followed by methylation-sensitive high-resolution melting.
Summary of Findings:
All 107 FFPE specimens yielded degraded DNA but the degree of degradation was highly variable among specimens. When specimens were categorized into three groups based on the degree of DNA degradation, those with the most intact DNA or in the intermediate group had higher spectrophotometric DNA yields than those that were the most degraded (P=0.0004 and P<0.0001, respectively). However, spectrophotometric yield did not always indicate specimen quality as some specimens demonstrated relatively intact DNA but low DNA yield and some specimens with high yields had very degraded DNA. Amplification of all three promoters occurred in specimens regardless of integrity, but the amplification curves shifted to the right with increasing degradation of the initial DNA despite starting with the same amount of bisulfite-modified DNA, indicating less amplifiable DNA in those samples. MS-HRM analysis was able to assess the methylation status of BRCA2 (not methylated) and miR-34a (methylated in many NSCLC specimens) promoters in all fifteen specimens examined. The methylation status of the APC promoter was determined in 14 of the 15 specimens (hypermethylated in some) with the exception being one of the five specimens with highly degraded DNA.
Biospecimens
Preservative Types
- Formalin
Diagnoses:
- Neoplastic - Carcinoma
Platform:
Analyte Technology Platform DNA Bisulfite conversion assay DNA Spectrophotometry DNA Real-time qPCR DNA Electrophoresis Pre-analytical Factors:
Classification Pre-analytical Factor Value(s) Real-time qPCR Specific Targeted nucleic acid APC
BRCA2
miR-34a
Spectrophotometry Specific Quality metrics Highly degraded DNA
Moderately degraded DNA
Less degraded DNA
Bisulfite conversion assay Specific Quality metrics Less degraded DNA
Moderately degraded DNA
Highly degraded DNA