DNA isolated from formalin-fixed paraffin-embedded healthy tissue after 30years of storage can be used for forensic studies.

Abstract

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 260nm and 280nm. The quality of isolated DNA was evaluated by PCR amplification of GPD1 (150bp), ACTB (262bp) and RPL4 (407bp) genes. The histomorphological characteristics of FFPE tissues were not significantly changed during 30years of storage. Higher yield (272.9 ± 10.3µg) 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 30years, the ACTB gene in the same samples up to 26years and the RPL4 gene up to 6years 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 30years. This is the first report using long-term archived healthy FFPE tissues (up to 30years) that shows that the DNA isolated from these tissues is of preserved integrity and can be used in molecular autopsy.