Impact of Quality and Quantity of DNA samples on KRAS Mutation Analysis
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Komkhum O, Wanapong D, Chuangsuwanich N, Mokkhamakkun C, Poungvarin N
Clinical Molecular Pathology Laboratory, Department pf Clinical Pathology, Faculty of Medicine Siriraj Hospital, Mahidol University, Bangkok, Thailand
Background: Many techniques based on polymerase chain reaction (PCR) have been utilized to detect KRAS mutation, a negative predictor of anti-EGFR treatment efficacy. Inaccurate mutation test can result from inefficient PCR amplification in poor quality DNA due to formalin-induced DNA degradation or the presence of PCR inhibitors. Objective: This study aims to investigate the overall quality of DNA isolated from Thai colorectal cancer specimens and appropriate DNA amount for detection of KRAS mutation.
Methods: We extracted DNA from 159 formalin-fixed paraffin-embedded (FFPE) tissues submitted to our laboratory for KRAS mutation analysis. Genomic DNA was measured by spectrophotometer. We utilized quantitative PCR technique to assess the quality of DNA. Different amounts of DNA were added in each PCR reaction to evaluate proper DNA quantity for amplification.