Formalin-fixed, paraffin-embedded (FFPE) tissue remains the preferred method of tissue preservation for clinical specimens. Compared to other methods of tissue preservation, FFPE samples are relatively stable at room temperature. This enables FFPE samples to be stored for long periods of time without the need for expensive refrigeration equipment or large amounts of facility space. For histological or morphological analyses, FFPE samples can be easily sectioned and stained to visualize both morphological and molecular features to identify disease states. Nucleic acids within the preserved tissue can be extracted and purified for downstream applications using PCR, quantitative PCR (qPCR) and next-generation sequencing (NGS). For these reasons and more, clinicians and researchers will likely continue to depend on the analysis of genetic material obtained from FFPE samples for years to come.
To use FFPE samples for molecular analysis, researchers are faced with several challenges. Formalin fixation, the process used to generate FFPE samples, is not standardized and can introduce greater variation across samples prepared by different clinicians or at different times. Studies conducted more than 20 years ago found that the condition of the tissue sample, mode of formalin use, and time of fixation can all affect the integrity of nucleic acids, which can compromise downstream analysis1. Furthermore, the buffers used during the fixation process can lead to the deamination of cytosine bases and single nucleotide modifications2. Fixation also induces cross-linking of nucleic acids with proteins as well as to other nucleic acids. To address cross-linking and deamination, samples must be treated in various ways to remove paraffin and reverse cross-linking while avoiding additional damage prior to nucleic acid extraction. These treatments, while necessary to obtain high-quality nucleic acid for molecular analysis, are time-consuming and do not address any fragmentation that may have occurred.
With an increasingly diverse set of genomic tools at their disposal, researchers and clinicians benefit from extracting as much nucleic acid as possible from each FFPE sample while still meeting the quality required for a more sensitive analysis method, such as NGS. While some nucleic acid extraction and purification technologies claim to be optimized for use with FFPE samples, they can further compromise the quality of the nucleic acids extracted and fail to extract all the nucleic acid available. These technologies also employ multi-step workflows that require optimal conditions at each step for successful extraction.
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