Multivariate Evaluation of DNA Quality Differences in Different Preanalytical Procedures in Mouse Livers.

Abstract

Successful histogenetic research relies on proper handling of tissue samples to maximize DNA quality. As the largest gland in the body, the liver is particularly sensitive to sample mishandling owing to its enzymatic and transcriptional activity. However, the impact of preanalytical procedures on the quality of extracted liver DNA remains poorly understood. In this study, we assessed the impact of extraction methods, duration of ex vivo liver ischemia, liver storage time, and temperature on extracted DNA quality. Comprehensive parameters such as DNA yields, purity, DNA integrity number, the percentage of double-stranded DNA (%dsDNA), and PCR amplification of the GAPDH gene fragment were assessed to identify the quality of extracted DNA. Our results revealed that these preanalytical processes had little effect on DIN values and PCR efficiency of GAPDH gene fragments for each sample, whereas the DNA yields, purity, and %dsDNAs varied widely across different processes. For liver DNA extraction, RNase is necessary to isolate "pure" DNA, and the presence of RNase could significantly increase the %dsDNA. In addition, significant increases in the yields, purity, and %dsDNA of extracted DNA were observed in the TissueLyser-processed livers compared with the mortar and pestle or shear cell disruption methods. Further investigation revealed that livers experiencing longer periods of ex vivo ischemia resulted in significantly compromised DNA yields, and to obtain sufficient DNA, the ex vivo liver ischemia should be limited to within 30 minutes. Moreover, compared with storage of livers at -80°C, storage of livers in the vapor phase of liquid nitrogen yielded a higher quality of the extracted DNA. Our findings exhibited significant implications for liver-derived DNA quality assessment and management.