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

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.

Keywords: DNA extraction; QIAamp kit; WaxFree DNA extraction kit; formalin-fixed and paraffin-embedded; quality check.

Figure 1.

The mean DNA yield per sample extracted from QIA and WAX. (A) A method based on UV absorbance at 260 nm. (B) Quantified using PicoGreen double-strand DNA-specific fluorescent dye. (C) Quantified using the quantitative polymerase chain reaction method. **P<0.01 vs. QIA. Data are presented as the mean ± standard deviations. QIA, QIAamp; WAX, WAXFree; UV, ultraviolet; Q41, the amount of DNA quantified using 41 bp primer pairs; Q129, the amount of DNA quantified using 129 bp primer pairs; Q305, the amount of DNA quantified using 305 bp primer pairs.

Figure 2.

Quality of extracted DNA. (A) An absorbance ratio of 260 to 280 nm. (B and C) Quantitative value ratio of each polymerase chain reaction product size, including Q129/Q41 and Q305/Q41. **P<0.01 vs. QIA. Data are presented as the mean ± standard deviations. QIA, QIAamp; WAX, WAXFree; Q41, the amount of DNA quantified using 41 bp primer pairs; Q129, the amount of DNA quantified using 129 bp primer pairs; Q305, the amount of DNA quantified using 305 bp primer pairs, A, absorbance.

Figure 3.

Age-related degradation of DNA. (A) The mean DNA yield per sample quantified by the UV method. (B) Quality of extracted DNA derived from each sample, quantified by an absorbance ratio of 260 to 280 nm. Quality of extracted DNA derived from each samples, quantified by qPCR with (C) Q129/Q41 and (D) Q305 and Q41. *P<0.05 vs. 0 years. ^#P<0.05 vs. 9 years. Data are presented as the mean ± standard deviations. UV, ultraviolet; qPCR, quantitative polymerase chain reaction; QIA, QIAamp; WAX, WAXFree; Q41, the amount of DNA quantified using 41 bp primer pairs; Q129, the amount of DNA quantified using 129 bp primer pairs; Q305, the amount of DNA quantified using 305 bp primer pairs, A, absorbance.