Dataset Information

Technical reproducibility of single-nucleotide and size-based DNA biomarker assessment using DNA extracted from formalin-fixed, paraffin-embedded tissues.

ABSTRACT: DNA extracted from formalin-fixed, paraffin-embedded (FFPE) tissues has been used in the past to analyze genetic polymorphisms. We evaluated the technical reproducibility of different types of assays for gene polymorphisms using DNA extracted from FFPE material. By using the MassARRAY iPLEX system, we investigated polymorphisms in DPYD (rs1801159 and rs3918290), UMPS (rs1801019), ERCC1 (rs11615), ERCC1 (rs3212986), and ERCC2 (rs13181) in 56 FFPE DNA samples. By using PCR, followed by size-based gel electrophoresis, we also examined TYMS 5' untranslated region 2R/3R repeats and GSTT1 deletions in 50 FFPE DNA samples and 34 DNAs extracted from fresh-frozen tissues and cell lines. Each polymorphism was analyzed by two independent runs. We found that iPLEX biomarker assays measuring single-nucleotide polymorphisms provided consistent concordant results. However, by using FFPE DNA, size-based PCR biomarkers (GSTT1 and TYMS 5' untranslated region) were discrepant in 32.7% (16/49, with exact 95% CI, 19.9%-47.5%; exact binomial confidence limit test) and 4.2% (2/48, with exact 95% CI, 0.5%-14.3%) of cases, respectively, whereas no discrepancies were observed using intact genomic DNA. Our findings suggest that DNA from FFPE material can be used to reliably test single-nucleotide polymorphisms. However, results based on size-based PCR biomarkers, and particularly GSTT1 deletions, using FFPE DNA need to be interpreted with caution. Independent repeated assays should be performed on all cases to assess potential discrepancies.

SUBMITTER: Zhang S

PROVIDER: S-EPMC4411247 | biostudies-literature | 2015 May

REPOSITORIES: biostudies-literature

ACCESS DATA

Publications

Technical reproducibility of single-nucleotide and size-based DNA biomarker assessment using DNA extracted from formalin-fixed, paraffin-embedded tissues.

Zhang Shenli S Tan Iain B IB Sapari Nur S NS Grabsch Heike I HI Okines Alicia A Smyth Elizabeth C EC Aoyama Toru T Hewitt Lindsay C LC Inam Imran I Bottomley Dan D Nankivell Matthew M Stenning Sally P SP Cunningham David D Wotherspoon Andrew A Tsuburaya Akira A Yoshikawa Takaki T Soong Richie R Tan Patrick P

The Journal of molecular diagnostics : JMD 20150304 3

DNA extracted from formalin-fixed, paraffin-embedded (FFPE) tissues has been used in the past to analyze genetic polymorphisms. We evaluated the technical reproducibility of different types of assays for gene polymorphisms using DNA extracted from FFPE material. By using the MassARRAY iPLEX system, we investigated polymorphisms in DPYD (rs1801159 and rs3918290), UMPS (rs1801019), ERCC1 (rs11615), ERCC1 (rs3212986), and ERCC2 (rs13181) in 56 FFPE DNA samples. By using PCR, followed by size-based ...[more]

PMID: 25746798

Similar Datasets

Project description:Formalin-fixed paraffin-embedded (FFPE) tissues are rarely used for screening DNA adducts of carcinogens because the harsh conditions required to reverse the formaldehyde-mediated DNA cross-links can destroy DNA adducts. We recently adapted a commercial silica-based column kit used in genomics to manually isolate DNA under mild conditions from FFPE tissues of rodents and humans and successfully measured DNA adducts of several carcinogens including aristolochic acid I (AA-I), 4-aminobiphenyl (4-ABP), and 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) (Yun et al. (2013) Anal. Chem. 85, 4251-8, and Guo et al. (2016) Anal. Chem. 88, 4780-7). The DNA retrieval methodology is robust; however, the procedure is time-consuming and labor intensive, and not amenable to rapid throughput processing. In this study, we have employed the Promega Maxwell 16 MDx system, which is commonly used in large scale genomics studies, for the rapid throughput extraction of DNA. This system streamlines the DNA isolation procedure and increases the sample processing rate by about 8-fold over the manual method (32 samples versus 4 samples processed per hour). High purity DNA is obtained in satisfactory yield for the measurements of DNA adducts by ultra performance liquid chromatography-electrospray-ionization-ion trap-multistage scan mass spectrometry. The measurements show that the levels of DNA adducts of AA-I, 4-ABP, and PhIP in FFPE rodent and human tissues are comparable to those levels measured in DNA from matching tissues isolated by the commercial silica-based column kits and in DNA from fresh frozen tissues isolated by the conventional phenol-chloroform extraction method. The isolation of DNA from tissues is one major bottleneck in the analysis of DNA adducts. This rapid throughput methodology greatly decreases the time required to process DNA and can be employed in large-scale epidemiology studies designed to assess the role of chemical exposures and DNA adducts in cancer risk.

Dataset Information

Technical reproducibility of single-nucleotide and size-based DNA biomarker assessment using DNA extracted from formalin-fixed, paraffin-embedded tissues.

Publications

Technical reproducibility of single-nucleotide and size-based DNA biomarker assessment using DNA extracted from formalin-fixed, paraffin-embedded tissues.

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets