DNA degrades during storage in formalin-fixed and paraffin-embedded tissue blocks

Abstract

Formalin-fixed paraffin-embedded (FFPE) tissue blocks are widely used to identify clinically actionable molecular alterations or perform retrospective molecular studies. Our goal was to quantify degradation of DNA occurring during mid to long-term storage of samples in usual conditions. We selected 46 FFPE samples of surgically resected carcinomas of lung, colon, and urothelial tract, of which DNA had been previously extracted. We performed a second DNA extraction on the same blocks under identical conditions after a median period of storage of 5.5 years. Quantitation of DNA by fluorimetry showed a 53% decrease in DNA quantity after storage. Quantitative PCR (qPCR) targeting KRAS exon 2 showed delayed amplification of DNA extracted after storage in all samples but one. The qPCR/fluorimetry quantification ratio decreased from 56 to 15% after storage (p < 0.001). Overall, remaining proportion of DNA analyzable by qPCR represented only 11% of the amount obtained at first extraction. Maximal length of amplifiable DNA fragments assessed with a multiplex PCR was reduced in DNA extracted from stored tissue, indicating that DNA fragmentation had increased in the paraffin blocks during storage. Next-generation sequencing was performed on 12 samples and showed a mean 3.3-fold decrease in library yield and a mean 4.5-fold increase in the number of single-nucleotide variants detected after storage. In conclusion, we observed significant degradation of DNA extracted from the same FFPE block after 4 to 6 years of storage. Better preservation strategies should be considered for storage of FFPE biopsy specimens.

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.…”

Overexpression of Sphingosine Kinase-1 and Sphingosine-1-Phosphate Receptor-3 in Severe Plasmodium falciparum Malaria with Pulmonary Edema

“…The present study has the following limitations. First, the specimens used in this study were lung tissues from autopsied malaria patients that had been stored for approximately 30 years and were thus unsuitable for determining the gene expression levels of SphK-1 and S1PR-3 via molecular techniques [35]. Second, it was not possible to determine the time course of SphK-1 and S1PR-3 expression during the malaria infection.…”

Real-Time PCR to Detect α-1 Antitrypsin S and Z Alleles in Formalin-Fixed Paraffin-Embedded Tissue

“…Given the age of this specimen (approximately 9 years), time-dependent DNA degradation likely impacted PCR amplification. A recent study demonstrated that, compared with DNA immediately extracted from FFPE tissue, archived samples stored for a mean of 5.4 years had 53% lower DNA quantity and only 11% of the original DNA could be amplified by real-time PCR (14). Time-dependent effects on DNA quality from biopsy specimens are predicted to be even greater.…”