Cold-temperature plastic resin embedding of liver for DNA- and RNA-based genotyping.
The standard practice of tissue fixation in 10% formalin followed by embedding in paraffin wax preserves cellular morphology at the expense of availability and quality of DNA and RNA. The negative effect on cellular constituents results from a combination of extensive cross-linking and strand scission of DNA, RNA, and proteins induced by formaldehyde as well as RNA loss secondary to ubiquitous RNase activity and negative effects of high temperature exposure during paraffin melting, microscopic section collection, and tissue adherence to glass slides. An effective strategy to correlate cellular phenotype with molecular genotype involves microdissection of tissue sections based on specific histopathological features followed by genotyping of minute representative samples for specific underlying molecular alterations. Currently, this approach is limited to short-length polymerase chain reaction amplification (<250 bp) of DNA, due to the negative effects of standard tissue fixation and processing. To overcome this obstacle and permit both cellular morphology and nucleic acid content to be preserved to the fullest extent, we instituted a system of cold-temperature plastic resin embedding based on the use of the water-miscible methyl methacrylate polymer known as Immunobed (Polysciences, Warminster, PA). The system is simple, easy to adapt to clinical practice, and cost-effective. Immunobed tissue sections demonstrate a cellular appearance equivalent or even superior to that of standard tissue sections. Moreover, thin sectioning (0.5-1.0 microm thickness) renders ultrastructural evaluation feasible on plastic-embedded blocks. Tissue microdissection is readily performed, yielding high levels of long DNA and RNA for genomic and transcription-based correlative molecular analysis. We recommend the use of Immunobed or similar products for use in molecular anatomical pathology.
Finkelstein
S D
SD
Department of Pathology, University of Pittsburgh Medical Center, Pennsylvania 15213, USA. finkelsteinsd@msx.upmc.edu
Dhir
R
R
Rabinovitz
M
M
Bischeglia
M
M
Swalsky
P A
PA
DeFlavia
P
P
Woods
J
J
Bakker
A
A
Becich
M
M
D003080:Cold Temperature; D004247:DNA / Q000235:genetics*; D004279:DNA, Viral / Q000235:genetics / Q000302:isolation & purification; D005838:Genotype; D016174:Hepacivirus / Q000235:genetics / Q000302:isolation & purification; D006801:Humans; D008099:Liver / Q000378:metabolism* / Q000821:virology; D016613:Plastic Embedding / Q000379:methods*; D012313:RNA / Q000235:genetics*; D012367:RNA, Viral / Q000235:genetics / Q000302:isolation & purification; D020133:Reverse Transcriptase Polymerase Chain Reaction
D004279:DNA, Viral; D012367:RNA, Viral; D012313:RNA; D004247:DNA