福尔马林固定石蜡包埋（FFPE）组织中的DNA降解对通过高分辨率熔解曲线分析（MS-HRM）进行的位点特异性甲基化评估的影响。

The influence of DNA degradation in formalin-fixed, paraffin-embedded (FFPE) tissue on locus-specific methylation assessment by MS-HRM.

作者信息

Daugaard Iben, Kjeldsen Tina E, Hager Henrik, Hansen Lise Lotte, Wojdacz Tomasz K

机构信息

Department of Biomedicine, Aarhus University, Wilhelm Meyers Allé 3, Build. 1230, DK-8000 Aarhus C, Denmark.

出版信息

Exp Mol Pathol. 2015 Dec;99(3):632-40. doi: 10.1016/j.yexmp.2015.11.007. Epub 2015 Nov 6.

DOI:10.1016/j.yexmp.2015.11.007

PMID:26551081

Abstract

Readily accessible formalin-fixed paraffin embedded (FFPE) tissues are a highly valuable source of genetic material for molecular analyses in both research and in vitro diagnostics but frequently genetic material in those samples is highly degraded. With locus-specific methylation changes being widely investigated for use as biomarkers in various aspects of clinical disease management, we aimed to evaluate to what extent standard laboratory procedures can approximate the quality of the DNA extracted from FFPE samples prior to methylation analyses. DNA quality in 107 FFPE non-small cell lung cancer (NSCLC) samples was evaluated using spectrophotometry and gel electrophoresis. Subsequently, the quality assessment results were correlated with the results of locus specific methylation assessment with methylation sensitive high resolution melting (MS-HRM). The correlation of template quality with PCR amplification performance and HRM based methylation detection indicated a significant influence of DNA quality on PCR amplification but not on methylation assessment. In conclusion, standard laboratory procedures fairly well approximate DNA degradation of FFPE samples and DNA degradation does not seem to considerably affect locus-specific methylation assessment by MS-HRM.

摘要

福尔马林固定石蜡包埋（FFPE）组织易于获取，是研究和体外诊断中分子分析的极有价值的遗传物质来源，但这些样本中的遗传物质经常高度降解。随着位点特异性甲基化变化在临床疾病管理的各个方面作为生物标志物的广泛研究，我们旨在评估标准实验室程序在多大程度上能够接近甲基化分析前从FFPE样本中提取的DNA质量。使用分光光度法和凝胶电泳评估了107个FFPE非小细胞肺癌（NSCLC）样本的DNA质量。随后，将质量评估结果与甲基化敏感高分辨率熔解（MS-HRM）的位点特异性甲基化评估结果进行关联。模板质量与PCR扩增性能以及基于HRM的甲基化检测之间的相关性表明，DNA质量对PCR扩增有显著影响，但对甲基化评估没有影响。总之，标准实验室程序相当好地近似了FFPE样本的DNA降解情况，并且DNA降解似乎不会对MS-HRM进行的位点特异性甲基化评估产生显著影响。