Methodology for preservation of high molecular-weight RNA in paraffin-embedded tissue: application for laser-capture microdissection.
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 hematoxylin-eosin 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.
Vincek
Vladimir
V
Department Pathology, University of Miami School of Medicine, Jackson Memorial Hospital, Holtz Center, Miami, Florida 33136, USA. vvincek@med.miami.edu
Nassiri
Mehdi
M
Block
Norman
N
Welsh
Catherine F
CF
Nadji
Mehrdad
M
Morales
Azorides R
AR
D019075:Electrophoresis, Capillary; D005404:Fixatives; D006416:Hematoxylin; D006801:Humans; D007834:Lasers; D000432:Methanol; D042282:Microdissection; D008872:Microwaves; D008970:Molecular Weight; D016612:Paraffin Embedding / Q000379:methods*; D011309:Preservation, Biological / Q000379:methods*; D012313:RNA / Q000032:analysis / Q000235:genetics / Q000302:isolation & purification*; D020133:Reverse Transcriptase Polymerase Chain Reaction
D005404:Fixatives; D012313:RNA; D000432:Methanol; D006416:Hematoxylin