Abstract
RNA and DNA analyses from paraffin-embedded tissues (PET) are an important diagnostic tool for characterization of a disease, exploring biomarkers and treatment options. Since nucleic acids from formalin-fixed and paraffin-embedded (FFPE) tissue are of limited use for molecular analyses due to chemical modifications of biomolecules alternate, formalin-free fixation reagents such as the PAXgene Tissue system are of evolving interest. Furthermore, biomedical research and biomarker development critically relies on using long-term stored PET from medical archives or biobanks to correlate molecular features with long-term disease outcomes. We therefore performed a comparative study to evaluate the effect of long term storage of FFPE and PAXgene Tissue-fixed and paraffin-embedded (PFPE) tissue at different temperatures on nucleic acid stability and usability in PCR. Matched FFPE and PFPE human tissues from routine clinical setting or rat tissues from a highly controlled animal model were stored at room temperature and 4°C, as well as in case of animal tissues frozen at -20°C and -80°C. RNA and DNA were extracted in intervals for up to nine years, and examined for integrity, and usability in quantitative RT-PCR (RT-qPCR) or PCR (qPCR) assays. PET storage at room temperature led to a degradation of nucleic acids which was slowed down by storage at 4°C and prevented by storage at -20°C or -80°C. Degradation was associated with an amplicon length depending decrease of RT-qPCR and qPCR efficiency. Storage at 4°C improved amplifiability in RT-qPCR and qPCR profoundly. Chemically unmodified nucleic acids from PFPE tissue performed superior compared to FFPE tissue, regardless of storage time and temperature in both human and rat tissues. In conclusion molecular analyses from PET can be greatly improved by using a non-crosslinking fixative and storage at lower temperatures such as 4°C, which should be considered in prospective clinical studies.
Highlights
Paraffin embedded tissue (PET) blocks are generated in huge quantities in the context of histopathological diagnostics and stored in large medical archives or biobanks
Thereby a correlation was found between RNA integrity and PET storage temperature for both, human tissues collected during routine clinical workflows (Fig 2) and rat tissue collected and processed under most stringently standardized preanalytical conditions (Fig 3)
As demonstrated here storage at room temperatures in routine archives leads to a loss of nucleic acid quality from PET blocks
Summary
Paraffin embedded tissue (PET) blocks are generated in huge quantities in the context of histopathological diagnostics and stored in large medical archives or biobanks. The funder QIAGEN GmbH provided support in the form of salaries for authors [DG, ND]. The specific roles of these authors are articulated in the ‘author contributions’ section. The funders did not have any additional role in the study design, data collection and analysis, decision to publish, or preparation of the manuscript.
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