Impact of storage conditions on the quality of nucleic acids in paraffin embedded tissues

• Published in: PloS one 2018-09, Vol.13 (9), p.e0203608-e0203608
• Main Authors: Groelz, Daniel, Viertler, Christian, Pabst, Daniela, Dettmann, Nadine, Zatloukal, Kurt
• Format: Article
• Language: English
• Subjects: Analysis; Animal models; Animal tissues; Animals; Archives & records; Bioindicators; Biological markers; Biology and Life Sciences; Biomarkers; Biomolecules; Comparative studies; Crosslinking; Cryopreservation; Degradation; Deoxyribonucleic acid; Diagnostic equipment (Medical); Diagnostic software; Diagnostic systems; DNA; DNA methylation; Fixatives - adverse effects; Fixatives - pharmacology; Formaldehyde; Gene expression; Genetic Markers; Health aspects; Human tissues; Humans; Low temperature; Medical treatment; Morphology; Nucleic acids; Nucleic Acids - analysis; Nucleic Acids - genetics; Organic chemistry; Paraffin; Paraffin Embedding; Paraffins; Pathology; Pets; Physical Sciences; Polymerase Chain Reaction; Prospective Studies; Proteins; Quality; R&D; Rats; Reagents; Research & development; Research and analysis methods; Ribonucleic acid; RNA; Storage; Storage conditions; Studies; Temperature; Temperature effects; Tissue Fixation; Tissue Preservation
• ISSN: 1932-6203; 1932-6203
• DOI: 10.1371/journal.pone.0203608

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.