An Optimized Method of Metabolite Extraction from Formalin-Fixed Paraffin-Embedded Tissue for GC/MS Analysis.
Formalin-fixed paraffin-embedded (FFPE) tissue specimens constitute a highly valuable source of clinical material for retrospective molecular studies. However, metabolomic assessment of such archival material remains still in its infancy. Hence, there is an urgent need for efficient methods enabling extraction and profiling of metabolites present in FFPE tissue specimens. Here we demonstrate the methodology for isolation of primary metabolites from archival tissues; either fresh-frozen, formalin-fixed or formalin-fixed and paraffin-embedded specimens of mouse kidney were analysed and compared in this work. We used gas chromatography followed by mass spectrometry (GC/MS approach) to identify about 80 metabolites (including amino acids, saccharides, carboxylic acids, fatty acids) present in such archive material. Importantly, about 75% of identified compounds were detected in all three types of specimens. Moreover, we observed that fixation with formalin itself (and their duration) did not affect markedly the presence of particular metabolites in tissue-extracted material, yet fixation for 24h could be recommended as a practical standard. Paraffin embedding influenced efficiency of extraction, which resulted in reduced quantities of several compounds. Nevertheless, we proved applicability of FFPE specimens for non-targeted GS/MS-based profiling of tissue metabolome, which is of great importance for feasibility of metabolomics studies using retrospective clinical material.
Center for Translational Research and Molecular Biology of Cancer, Maria Sklodowska-Curie Memorial Cancer Center and Institute of Oncology, Gliwice Branch, Wybrzeze Armii Krajowej 15, 44-100, Gliwice, Poland.
Anna
A
Wojakowska
Institute of Bioorganic Chemistry Polish Academy of Sciences, Noskowskiego 12/14, 61-704 Poznan, Poland.
Łukasz
Ł
Marczak
Center for Translational Research and Molecular Biology of Cancer, Maria Sklodowska-Curie Memorial Cancer Center and Institute of Oncology, Gliwice Branch, Wybrzeze Armii Krajowej 15, 44-100, Gliwice, Poland.
Karol
K
Jelonek
Warwick Systems Biology Centre, University of Warwick, Coventry, CV4 7AL, United Kingdom.
Krzysztof
K
Polanski
Center for Translational Research and Molecular Biology of Cancer, Maria Sklodowska-Curie Memorial Cancer Center and Institute of Oncology, Gliwice Branch, Wybrzeze Armii Krajowej 15, 44-100, Gliwice, Poland.
Piotr
P
Widlak
Center for Translational Research and Molecular Biology of Cancer, Maria Sklodowska-Curie Memorial Cancer Center and Institute of Oncology, Gliwice Branch, Wybrzeze Armii Krajowej 15, 44-100, Gliwice, Poland.
Monika
M
Pietrowska
D000596:Amino Acids / Q000378:metabolism*; D000818:Animals; D002264:Carboxylic Acids / Q000378:metabolism*; D005227:Fatty Acids / Q000378:metabolism*; D005557:Formaldehyde / Q000737:chemistry; D008401:Gas Chromatography-Mass Spectrometry / Q000379:methods; D055442:Metabolome; D051379:Mice; D010232:Paraffin / Q000737:chemistry; D016612:Paraffin Embedding; D016707:Tissue Fixation
D000596:Amino Acids; D002264:Carboxylic Acids; D005227:Fatty Acids; D005557:Formaldehyde; D010232:Paraffin