Faculty of Medical Sciences, Department of Forensic Medicine, University of Kragujevac, Kragujevac, Serbia.
Institute of Forensic Medicine, Faculty of Medicine, University of Belgrade, Belgrade, Serbia.
Forensic Sci Med Pathol. 2021 Mar;17(1):47-57. doi: 10.1007/s12024-020-00327-z. Epub 2020 Nov 7.
Tissue formalin fixation and paraffin embedding (FFPE) is a standard method for long-term preservation and morphological and molecular analysis. The aim of this study was to analyze the effect of storage time on the integrity of DNA isolated from three different healthy FFPE tissues. DNA was isolated from FFPE heart, liver and brain tissues obtained from autopsy and archived from 1988 to 2017 using two different methods of DNA isolation: phenol-chloroform-isoamyl alcohol (PCI) and PureLink Genomic DNA Kit. The quantification and purity of DNA was measured spectrophotometrically at 260 nm and 280 nm. The quality of isolated DNA was evaluated by PCR amplification of GPD1 (150 bp), ACTB (262 bp) and RPL4 (407 bp) genes. The histomorphological characteristics of FFPE tissues were not significantly changed during 30 years of storage. Higher yield (272.9 ± 10.3 µg) and purity (A260/280 = 2.05) of DNA was obtained using the PCI method for DNA isolation from FFPE liver tissue. The PCI extraction method showed reproducible and consistent results in PCR amplification of all of three examined genes. The GPD1 gene can be amplified up to 30 years, the ACTB gene in the same samples up to 26 years and the RPL4 gene up to 6 years of storage in FFPE blocks. Although the best yield and purity of isolated DNA (using both isolation methods) was obtained from FFPE liver tissue, the DNA with the most preserved integrity was obtained from brain tissue archived up to 30 years. This is the first report using long-term archived healthy FFPE tissues (up to 30 years) that shows that the DNA isolated from these tissues is of preserved integrity and can be used in molecular autopsy.
组织福尔马林固定和石蜡包埋（FFPE）是长期保存和形态学及分子分析的标准方法。本研究旨在分析储存时间对从三种不同健康 FFPE 组织中分离的 DNA 完整性的影响。使用两种不同的 DNA 分离方法：酚-氯仿-异戊醇（PCI）和 PureLink 基因组 DNA 试剂盒，从 1988 年至 2017 年从尸检获得并归档的 FFPE 心脏、肝脏和脑组织中分离 DNA。使用分光光度法在 260nm 和 280nm 处测量 DNA 的定量和纯度。通过 PCR 扩增 GPD1（150bp）、ACTB（262bp）和 RPL4（407bp）基因评估分离 DNA 的质量。在 30 年的储存过程中，FFPE 组织的组织形态学特征没有明显变化。使用 PCI 方法从 FFPE 肝组织中分离 DNA 时，获得了更高的产量（272.9±10.3μg）和纯度（A260/280=2.05）。PCI 提取方法在所有三个检测基因的 PCR 扩增中均显示出可重复和一致的结果。GPD1 基因可在 FFPE 块中扩增长达 30 年，ACTB 基因在相同样本中可扩增长达 26 年，RPL4 基因可扩增长达 6 年。尽管使用两种分离方法从 FFPE 肝组织中获得了最佳产量和纯度的 DNA，但从存档长达 30 年的脑组织中获得了保存最完整的 DNA。这是首次使用长达 30 年的长期存档健康 FFPE 组织（长达 30 年）的报告，表明从这些组织中分离的 DNA 具有保存完整的完整性，可用于分子尸检。
