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Sci Rep. 2018 Apr 20;8(1):6351. doi: 10.1038/s41598-018-24781-6.

Systematic evaluation of RNA quality, microarray data reliability and pathway analysis in fresh, fresh frozen and formalin-fixed paraffin-embedded tissue samples.

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Department of Neuroimmunology, Center for Brain Research, Medical University of Vienna, Vienna, Austria.
Department of Neuroimmunology, Center for Brain Research, Medical University of Vienna, Vienna, Austria.
Ann Romney Center for Neurological Diseases, Brigham and Women's Hospital, Harvard Medical School, Boston, USA.
Epilepsy Center Bethel, Krankenhaus Mara, Bielefeld, Germany.


Formalin-fixed paraffin-embedded (FFPE) tissues are valuable resources commonly used in pathology. However, formalin fixation modifies nucleic acids challenging the isolation of high-quality RNA for genetic profiling. Here, we assessed feasibility and reliability of microarray studies analysing transcriptome data from fresh, fresh-frozen (FF) and FFPE tissues. We show that reproducible microarray data can be generated from only 2 ng FFPE-derived RNA. For RNA quality assessment, fragment size distribution (DV200) and qPCR proved most suitable. During RNA isolation, extending tissue lysis time to 10 hours reduced high-molecular-weight species, while additional incubation at 70 °C markedly increased RNA yields. Since FF- and FFPE-derived microarrays constitute different data entities, we used indirect measures to investigate gene signal variation and relative gene expression. Whole-genome analyses revealed high concordance rates, while reviewing on single-genes basis showed higher data variation in FFPE than FF arrays. Using an experimental model, gene set enrichment analysis (GSEA) of FFPE-derived microarrays and fresh tissue-derived RNA-Seq datasets yielded similarly affected pathways confirming the applicability of FFPE tissue in global gene expression analysis. Our study provides a workflow comprising RNA isolation, quality assessment and microarray profiling using minimal RNA input, thus enabling hypothesis-generating pathway analyses from limited amounts of precious, pathologically significant FFPE tissues.

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