Methodology for preservation of high molecular-weight RNA in paraffin-embedded tissue: Application for laser-capture microdissection

Vladimir Vincek, Mehdi Nassiri, Norman Block, Catherine F. Welsh, Mehrdad Nadji, Azorides R. Morales

Research output: Contribution to journalArticle

28 Scopus citations

Abstract

Laser-capture microdissection techniques have enhanced the ability to perform molecular studies of pure-cell populations. Although many technical factors affect the outcome of the procedure, none is more critical than the appropriate handling of the tissue. Because extraction of intact RNA from paraffin-embedded tissue is a difficult and inconsistent process, frozen sections with their attendant problems are used for this purpose. The major limitation of frozen section is its inferior morphologic quality compared with paraffin-embedded sections that may complicate accurate identification of cells during microdissection. We have developed a procedure that provides both high-quality histomorphology and RNA preservation in paraffin-embedded tissue. It is based on the use of a methanol-based fixative coupled with microwave-assisted rapid tissue processing. This technology in conjunction with a modified hematoxylineosin stain and a RNA extraction method allows isolation of high molecular-weight RNA from laser-capture microdissected, hematoxylin and eosin-stained paraffin sections. The high quality of the extracted RNA was confirmed by capillary electrophoresis and RT-PCR. The combination of a methanol-based fixative, rapid microwave tissue processing, and a modified hematoxylin and eosin stain produces paraffin sections that yield high molecular-weight RNA upon microdissection. This methodology opens the door for a wide range of gene expression analyses using paraffin-embedded tissue.

Original languageEnglish (US)
Pages (from-to)127-133
Number of pages7
JournalDiagnostic Molecular Pathology
Volume14
Issue number3
DOIs
StatePublished - Sep 1 2005

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Keywords

  • Laser-capture microdissection
  • Paraffin-embedded tissue
  • RNA

ASJC Scopus subject areas

  • Pathology and Forensic Medicine

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