Nucleic acids from long-term preserved FFPE tissues are suitable for downstream analyses

Abstract

Tissues used for clinical diagnostics are mostly formalin-fixed and paraffin-embedded (FFPE) which provides many advantages. However, the quality of the obtained nucleic acids (NA) is reduced and this turns out to be a challenge for further molecular analyses. Although the spectrum of analyses of NA extracted from FFPE tissue has increased, the standard operating procedures for NA isolation from old tissue blocks still need to be improved. Here, we compared the efficiency of different NA extraction methods, using FFPE tissues of variable age and origin, with respect to downstream analyses. Our study showed that the phenol-chloroform isoamyl alcohol (PCI) and the commercial Qiagen protocol yielded samples with highest purity. The PCI protocol delivered the longest amplicons even from samples from the 1970s. We developed a short (1 h) tissue lysis procedure that turned out to be highly time- and cost-effective when DNA quality was tested using single and multiplex PCR. Compared to a 1-day lysis-protocol, the amplicons were only 100 bp shorter. In addition, single-copy genes used in daily routine were successfully amplified from long-term stored FFPE samples following 1-h tissue-lysis. The RNA integrity numbers (RIN) determined on RNA isolated from FFPE tissues indicated degraded RNA; however, all RINs were above the generally agreed threshold of 1.4. We showed that, depending on the purpose of the analysis, NA retrieved from FFPE tissues older than 40 years may be successfully used for molecular analysis.

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

“…Several studies have previously evaluated the integrity of DNA from FFPE tissues or compared methods for DNA extraction from FFPE tissues (8,(11)(12)(13)(14)(15); however, the majority of samples used in previous studies were subject to strictly controlled fixation methods and storage conditions. In the present study, the age-related degradation of DNA was assessed using various methods (UV, FL and qPCR), for FFPE tissues stored in a normal clinical hospital.…”

Sequence Artifacts in DNA from Formalin-Fixed Tissues: Causes and Strategies for Minimization

“…Fragmentation of DNA in formalin-fixed tissues was shown to be increased with longer storage time and lower pH of formalin used in tissue fixation (29 ). Compared to DNA from fresh formalin-fixed tissues, the PCR success rate of DNA from older formalin-fixed tissues was shown to be decreased (29 ), indicating that DNA fragmentation may continuously occur during storage. Fragmentation damage in FFPE DNA directly influences the amount of templates available for PCR amplification (30 ).…”

Optimizing Fixation Protocols to Improve Molecular Analysis from FFPE Tissues

“…Concerning the post-fixation steps (temperature and duration of FPPE block storage), there is some controversy in the literature whether storage of paraffin blocks under controlled conditions of temperature may prevent DNA degradation (13). The lack of studies on this topic and the different measurement protocols of nucleic acid used, some of them not comparable, may be the cause of this unsolved problem (6).…”

“…Despite the many advantages there are in formalin fixation of tissue samples for diagnostic purposes, use of FFPE material for molecular analysis remains problematic (13). Formaldehyde as a 10% neutral buffered formalin is the most used fixative because it preserves an extensive range of tissues and tissue components, and considerably inexpensive as well.…”