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Systematic evaluation of RNA quality, microarray data reliability and pathway analysis in fresh, fresh frozen and formalin-fixed paraffin-embedded tissue samples.
Wimmer, Isabella; Tröscher, Anna R; Brunner, Florian; Rubino, Stephen J; Bien, Christian G; Weiner, Howard L; Lassmann, Hans; Bauer, Jan.
  • Wimmer I; Department of Neuroimmunology, Center for Brain Research, Medical University of Vienna, Vienna, Austria. isabella.wimmer@meduniwien.ac.at.
  • Tröscher AR; Department of Neuroimmunology, Center for Brain Research, Medical University of Vienna, Vienna, Austria.
  • Brunner F; Department of Neuroimmunology, Center for Brain Research, Medical University of Vienna, Vienna, Austria.
  • Rubino SJ; Ann Romney Center for Neurological Diseases, Brigham and Women's Hospital, Harvard Medical School, Boston, USA.
  • Bien CG; Epilepsy Center Bethel, Krankenhaus Mara, Bielefeld, Germany.
  • Weiner HL; Ann Romney Center for Neurological Diseases, Brigham and Women's Hospital, Harvard Medical School, Boston, USA.
  • Lassmann H; Department of Neuroimmunology, Center for Brain Research, Medical University of Vienna, Vienna, Austria.
  • Bauer J; Department of Neuroimmunology, Center for Brain Research, Medical University of Vienna, Vienna, Austria.
Sci Rep ; 8(1): 6351, 2018 04 20.
Article en En | MEDLINE | ID: mdl-29679021
ABSTRACT
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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Texto completo: 1 Banco de datos: MEDLINE Asunto principal: ARN / Reproducibilidad de los Resultados / Análisis de Secuencia de ARN Tipo de estudio: Prognostic_studies Límite: Animals / Humans Idioma: En Año: 2018 Tipo del documento: Article

Texto completo: 1 Banco de datos: MEDLINE Asunto principal: ARN / Reproducibilidad de los Resultados / Análisis de Secuencia de ARN Tipo de estudio: Prognostic_studies Límite: Animals / Humans Idioma: En Año: 2018 Tipo del documento: Article