Abstract
Precise quantitation of protein biomarkers in clinical tissue specimens is a prerequisite for accurate and effective diagnosis, prognosis, and personalized medicine. Although progress is being made, protein analysis from formalin-fixed and paraffin-embedded tissues is still challenging. In previous reports, we showed that the novel formalin-free tissue preservation technology, the PAXgene Tissue System, allows the extraction of intact and immunoreactive proteins from PAXgene-fixed and paraffin-embedded (PFPE) tissues. In the current study, we focused on the analysis of phosphoproteins and the applicability of two-dimensional gel electrophoresis (2D-PAGE) and enzyme-linked immunosorbent assay (ELISA) to the analysis of a variety of malignant and non-malignant human tissues. Using western blot analysis, we found that phosphoproteins are quantitatively preserved in PFPE tissues, and signal intensities are comparable to that in paired, frozen tissues. Furthermore, proteins extracted from PFPE samples are suitable for 2D-PAGE and can be quantified by ELISA specific for denatured proteins. In summary, the PAXgene Tissue System reliably preserves phosphoproteins in human tissue samples, even after prolonged fixation or stabilization times, and is compatible with methods for protein analysis such as 2D-PAGE and ELISA. We conclude that the PAXgene Tissue System has the potential to serve as a versatile tissue fixative for modern pathology.
Highlights
For accurate diagnosis of diseases such as cancer, histopathological analysis is still the gold standard
We evaluated the applicability of proteins extracted from PAXgene-fixed and paraffin-embedded tissues (PFPE) tissues for twodimensional gel electrophoresis (2D-PAGE) and enzyme-linked immunosorbent assays (ELISA)
To gain insights into the capability of the PAXgene Tissue System to preserve the phosphoproteome, in the current study we investigated different human malignant (n = 3) and non-malignant (n = 17) tissues with particular emphasis on phosphoproteins
Summary
For accurate diagnosis of diseases such as cancer, histopathological analysis is still the gold standard. Molecular diagnostics and the elucidation of the mechanistic pathways of malignancy have been incorporated into routine pathology for the purpose of stratifying cancer patients for the most effective individualized therapy Crucial to these personalized approaches to therapy and for biomarker discovery programs is the analysis of high quality clinical tissue specimens which are suitable for conventional histopathological diagnosis as well as accurate detection and quantification of nucleic acids and proteins. Improvements of the standard formalin fixation process, such as formalin fixation at low temperature, demonstrate a better preservation of the integrity of nucleic acids [8] This approach improves the preservation of the molecular content of FFPE tissue but, the level of nucleic acid quality obtained from cryopreserved tissue samples is not reached. More important is the fact that PAXgene-fixed and paraffin-embedded tissues (PFPE) provide nucleic acid and protein quantity and quality similar to that found in cryopreserved tissue samples
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