Formalin Fixation of Human Healthy Autopsied Tissues: The Influence of Type of Tissue, Temperature and Incubation Time on the Quality of Isolated DNA

Abstract

Abstract Formalin fixation is a widely used method in histopathology that has certain limits. Formalin often leads to the degradation of DNA molecules in cancer tissues, which makes tissues unusable for molecular analysis. The other factors may also affect the quality of DNA isolated from fixed tissues. The aim of this study is to determine the impact of the incubation time and temperature on the quality of DNA molecules isolated from various healthy human tissues. The brain, lung and kidney tissues, excluded during the forensic autopsies of people who died of violent death, were fixed in phosphate-buffered formalin from 24h to two months. After the completion of the incubation period, the DNA was isolated using phenol-chloroform-isoamyl alcohol extraction method and the concentration and purity of the samples were determined spectrophotometrically. The degree of degradation of DNA was assessed by PCR reaction, by amplification of gene fragments which lengths were 150bp (GPD1) and 262bp (β-actin). The highest concentration, purity and preserved integrity of DNA were obtained from the brain samples. With prolonged tissue incubation times in formalin, the concentration and integrity of DNA decreased in all tissue samples, especially in the brain tissue, while the purity of DNA remained unchanged. Also, tissue fixation at +4°C contributed to a better quality of isolated DNA compared to DNA isolated from tissue fixed at room temperature. We can conclude that the type of human healthy tissue, temperature and the incubation time of formalin fixation have important influence on the concentration, purity and integrity of DNA during fixation of tissues excluded in the course of forensic autopsy.