Technical Reproducibility of Single-Nucleotide and Size-Based DNA Biomarker Assessment Using DNA Extracted from Formalin-Fixed, Paraffin-Embedded Tissues

Publication date: Available online 4 March 2015 Source:The Journal of Molecular Diagnostics Author(s): Shenli Zhang , Iain BeeHuat Tan , Nur S. Sapari , Heike Grabsch , Alicia Okines , Elizabeth C. Smyth , Toru Aoyama , Lindsay C. Hewitt , Imran Inam , Dan Bottomley , Matthew Nankivell , Sally P. Stenning , David Cunningham , Andrew Wotherspoon , Akira Tsuburaya , Takaki Yoshikawa , Richie Soong , Patrick Tan 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) ...

Source: The Journal of Molecular Diagnostics - March 8, 2015 Category: Pathology Source Type: research