Cold-Temperature Plastic Resin Embedding of Liver for DNA- and RNA-Based Genotyping

Abstract

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.

Hadp1, a newly identified pleckstrin homology domain protein, is required for cardiac contractility in zebrafish

“…TEM was performed as previously described ( Jurynec et al, 2008 ). For standard histology, 48-hpf embryos were fixed in 4% PFA overnight, dehydrated and processed as described ( Finkelstein et al, 1999 ). 5-μm sections were stained with Gill’s hematoxylin (Sigma) and counterstained with an alcoholic eosin Y solution (Shandon).…”

Comparison of short-term and long-term protocols for stabilization and preservation of RNA and DNA of Leishmania, Trypanosoma, and Plasmodium

“…Of the nucleic acids, DNA is relatively stable while RNA is fairly labile and will degrade easily in routine conditions. Therefore, the molecular analysis of RNA in particular is severely hampered as a result of loss from abundant and stable tissue RNAses and the high temperatures commonly used in specimen processing (Finkelstein et al, 1999). To circumvent this problem, the RNA in pathological specimens is preserved through removal or inactivation of any RNAses present in a biological specimen by addition of commercial products or chemicals (Fregeau et al, 2001).…”

Low‐temperature glycol methacrylate resin embedding method: A protocol suitable for bone marrow immunohistochemistry, PCR, and fish analysis

“…Glycol methacrylate (GMA) embedding of BM offers several advantages (Church et al,1997; Finkelstein et al,1999; Gaiser and Bernhards,2007; Ogborn and Sareen,1995) as follows: (1) cold embedding procedure may protect heat‐sensitive antigens better; (2) excellent preservation of cellular morphology by the plastic sections and hence provide good correlation of morphologic characteristics with marker reactions; (3) novel GMA resin embedding techniques preserve immunoreactivity as well as amplifiable DNA and mRNA as an alternative to paraffin embedding; and (4) GMA resin is nonfluorescent, thus suitable for immunofluorescence or FISH technique.…”