Brain Pathology, Sept, 2012, p.(1)
To purchase or authenticate to the full-text of this article, please visit this link: http://onlinelibrary.wiley.com/doi/10.1111/j.1750-3639.2012.00564.x/abstract Byline: Sylvia Eisele(1)(2), Markus Krumbholz(1)(2), Marie-Therese Fischer(3), Hema Mohan(2), Andreas Junker(1)(2)(*), Thomas Arzberger(4), Reinhard Hohlfeld(1)(2), Monika Bradl(3), Hans Lassmann(3), Edgar Meinl(1)(2) Keywords: FFPE; microRNA; molecular analysis; multiple sclerosis; quantitative PCR; transcriptome Abstract The elaboration of novel pathogenic aspects of multiple sclerosis (MS) requires the analysis of well-defined stages of lesion development. However, specimens of certain stages and lesion types are either present in small brain biopsies, insufficient in size for further molecular studies or available as formalin-fixed and paraffin-embedded (FFPE) material only. Therefore, application of current molecular biology techniques to FFPE tissue is warranted. We compared FFPE and frozen tissue by using quantitative polymerase chain reaction and report: (1) FFPE material is highly heterogeneous regarding the utility for transcript profiling of mRNAs; well-preserved FFPE samples had about a 100-fold reduced sensitivity compared with frozen tissue, but gave similar results for genes of sufficient abundance; (2) FFPE samples not suitable for mRNA analysis are still highly valuable for miRNA quantification; (3) the length of tissue fixation greatly affects utility for mRNA but not for miRNA analysis; (4) FFPE samples can be processed via a hot water bath for dissection of defined lesion areas; and (5) in situ hybridization for proteolipid protein (PLP) helps to identify samples not suitable for mRNA amplification. In summary, we present a detailed protocol how to use autoptic FFPE tissue for transcript profiling in dissected tissue areas. Author Affiliation: (1)Institute of Clinical Neuroimmunology, University Hospital Gro[sz]hadern, Ludwig-Maximilians-University, Munich (2)Department of Neuroimmunology, Max-Planck-Institute of Neurobiology, Martinsried, Germany (3)Center for Brain Research, Medical University of Vienna, Vienna, Austria (4)Center for Neuropathology and Prion Research, Ludwig-Maximilians-University, Munich, Germany Correspondence: (*) Edgar Meinl, MD, Department of Neuroimmunology, Max-Planck-Institute of Neurobiology, Am Klopferspitz 18, 82152 Martinsried, Germany (E-mail: firstname.lastname@example.org) Article Note: (*) Present address: Institute of Neuropathology, University Medical Center, Robert-Koch-Str. 40, 37099 Gottingen, Germany. ([dagger]) Grants and Support: The Deutsche Forschungsgemeinschaft (SFB 571), Herrmann and Lilly Schilling Foundation, Verein zur Therapieforschung fur Multiple-Sklerose-Kranke, the Bundesministerium fur Bildung und Forschung (aKrankheitsbezogenes Kompetenznetz Multiple Sklerosea), the Excellency Initiative of the Ludwig Maximilians University Munich and the scholarship program aForderung fur Forschung und Lehre (FoFoLe)a of the Ludwig Maximilians University Munich, Germany. Received 29 September 2011; Accepted 28 November 2011; Published Online Article Accepted 10 January 2012 Supporting information: Additional Supporting Information may be found in the online version of this article Table S1. Analyzed tissue specimens. Table S2. Extracellular matrix and extracellular matrix related genes and corresponding TaqManA[R] PCR assays used for TaqManA[R] Low Density Array (LDA) analysis (Applied Biosystems, Darmstadt, Germany). Table S3. Protocol for using archival FFPE specimen.
Messenger Rna -- Analysis ; Scholarships (Financial aid) -- Analysis ; Medical Schools -- Analysis ; Genes -- Analysis ; Microrna -- Analysis ; Multiple Sclerosis -- Analysis
Cengage Learning, Inc.