Estimation of age-related DNA degradation from formalin-fixed and paraffin-embedded tissue according to the extraction methods

Abstract

Abstract.Techniques for the extraction and use of nucleic acids from formalin-fixed and paraffin-embedded (FFPE) tissues, preserved over long time periods in libraries, have been developed. However, DNA extracted from FFPE tissues is generally damaged, and long-term storage may affect DNA quality. Therefore, it is important to elucidate the effect of long-term storage on FFPE tissues and evaluate the techniques used to extract DNA from them. In the present study, the yield, purity, and integrity of DNA in FFPE tissue samples was evaluated. Two DNA extraction techniques were used: A silica-binding DNA collection method using QIAamp DNA FFPE Tissue kit (QIA) and a total tissue DNA collection method using a WaxFree DNA extraction kit (WAX). A total of 25 FFPE tissues from lung adenocarcinomas were studied, which had been surgically resected and fixed at Okayama University Hospital prior to examination and subsequent storage at room temperature for 0.5, 3, 6, 9 and 12 years. Extracted DNA was quantified using ultraviolet absorbance, fluorescent dye, and quantitative polymerase chain reaction (qPCR). The quality of the DNA was defined by the absorbance ratio of 260 to 280 nm (A260/280) and Q-score, which is the quantitative value of qPCR product size ratio. The results demonstrated that the yield of total DNA extracted using WAX was significantly greater than when QIA was used (P<0.01); however, DNA extracted using WAX included more contaminants and was significantly more fragmented compared with DNA extracted using QIA (P<0.01). Aging had no significant effect on absolute DNA yield or DNA purity, although it did significantly contribute to increased DNA degradation for both QIA and WAX extraction (QIA P=0.02, WAX P=0.03; 0.5 years vs. 3 years, QIA P<0.01, WAX P=0.03; 9 years vs. 12 years). Both extraction methods are viable depending on whether high yield or high quality of extracted DNA is required. However, due to the increased degradation with age, storage time limits the available DNA in FFPE tissues regardless of the extraction method.

Association Between Preanalytical Factors and Tumor Mutational Burden Estimated by Next-Generation Sequencing-Based Multiplex Gene Panel Assay

“…These observations corroborated with those reported in previous studies demonstrating that DNA degradation is more evident in FFPE samples than in FF samples owing to the fragmentation and chemical modification of DNA . The sample storage period also reportedly influences DNA degradation in FFPE samples . In the current study, the sample storage period was inversely correlated with the DNA library concentration (r = ‐0.4, p < .01; supplemental online Fig.…”

Evaluation of commercial DNA and RNA extraction methods for high-throughput sequencing of FFPE samples

“…However, high concordance has been reported between paired fresh frozen and FFPE samples in molecular analyses [ 1 ]. The nucleic acid quality and integrity are affected by the pH of the fixative, the length of tissue fixation, the age and storage condition of tissue blocks and the extraction method used [ 2 – 4 ]. Formaldehyde, the active component of formalin, chemically cross-links the nucleic acids with the surrounding proteins and may also modify the nucleotides.…”

Differentiation of lymphocytic‐plasmacytic enteropathy and small cell lymphoma in cats using histology‐guided mass spectrometry

“…Finally, samples used in this study were surplus FFPE samples stored in the tissue archive of the of the Texas A&M Gastrointestinal Laboratory. With increasing storage time, DNA quality decreases 38 and might have affected results of the clonality assay.…”