The changes in tissue histomorphology and quality of DNA in healthy human autopsied tissues stored at -20 °C and -150 °C.

ABSTRACT

The aim of this study was to compare the changes in tissue histomorphology and DNA quality in six different healthy tissues (brain, heart, lung, liver, spleen and kidney) exempted during autopsy of healthy individuals and storage at -20 °C and -150 °C three month. Tissue samples were obtained, divided by tissue and temperature group, and for each sample, tissue histomorphology and DNA (isolated from all tissues in duplicated - 72 samples of DNA) quality were analysed. Morphology of tissue samples was studied using H&E staining. DNA was isolated using the phenol-chloroform-isoamyl alcohol method. To assess the concentration and purity of the DNA samples, we used a spectrophotometer to measure absorbance at wavelengths of 280 nm and 260 nm. The fragments of human telomerase reverse transcriptase (hTERT) gene were amplified from the DNA using PCR reaction and then visualised using the 2 % agarose gel. Samples stored at -150 °C sustained the highest degree of histomorphological damage, while samples stored at -20 °C were less degraded, compared to control. The liver samples stored at -20 °C had a mean DNA concentration (1030.4 ± 51.5 ng/µl) higher than the samples of liver tissue stored at -150 °C (497.4 ± 167.1 ng/µl) (p < 0.001). Other tissues did not have statistically significantly different DNA concentration at both temperatures. Liver samples at -20 °C had degraded DNA, showed as the absence of hTERT gene in most of samples. Other tissue samples in both temperature groups had unfragmented DNA. Storing tissue samples at -20 °C is not inferior in terms of DNA yield and integrity, and possibly superior for tissue histomorphology, comparing with samples stored at -150°C.