Optimized RNA extraction from non-deparaffinized, laser-microdissected material

• Published in: Methods in molecular biology (Clifton, N.J.) Vol. 755; p. 67
• Main Authors: Jonigk, Danny, Modde, Friedrich, Bockmeyer, Clemens L, Becker, Jan Ulrich, Lehmann, Ulrich
• Format: Journal Article
• Language: English
• Published: United States 01.01.2011
• Subjects: Cell Separation - methods; Data Interpretation, Statistical; Gene Expression; Gene Expression Profiling - methods; Genes, Wilms Tumor; Humans; Kidney Glomerulus - cytology; Kidney Glomerulus - metabolism; Lasers; Membrane Proteins - genetics; Membrane Proteins - metabolism; Microdissection - methods; Microtomy - methods; Paraffin Embedding; Pulmonary Veins - cytology; Reverse Transcriptase Polymerase Chain Reaction - methods; RNA Polymerase II - genetics; RNA Polymerase II - metabolism; RNA, Messenger - genetics; RNA, Messenger - isolation & purification; Tissue Fixation - methods; Umbilical Arteries - cytology
• ISSN: 1940-6029
• DOI: 10.1007/978-1-61779-163-5_5

mRNA extraction and subsequent RT-polymerase chain reaction (PCR)-based expression analysis from laser-microdissected material is by now a well-established and reproducible method. Most routinely stored tissue samples are preserved as formalin-fixed, paraffin-embedded materials. While this allows for a convenient storage and stable preservation of nucleic acids, deparaffinization before staining for laser microdissection may result in a significant loss of mRNA quality and consequently of PCR sensitivity. We describe a method of isolating anatomic compartments from non-deparaffinized, formalin-fixed, and paraffin-embedded tissues by laser-assisted microdissection which allows for a highly efficient mRNA retrieval.