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The Korean Journal of Pathology 2001;35(2): 98-110.
Improved Technique of Digoxigenin Labeled RNA in situ Hybridization.
Suk Keun Lee, Yeon Sook Kim, In Sun Song, Sang Shin Lee, Young Jun Lee, Woo Ho Kim, Je Geun Chi
1Department of Oral Pathology, Kangnung National University Dental College, Kangnung 210-711, Korea. sklee@knusun.kangnung.ac.kr
2Department of Pathology, Seoul National University College of Medicine, Seoul 110-799, Korea.
BACKGROUND: A practical RNA in situ hybridization method using digoxigenin labeled RNA probes is described in order to evaluate the technical difficulties and problems in RNA in situ hybridization. METHODS: The paraffin sections, routinely processed in the Pathology Laboratory, were tested for the possibility of RNA in situ hybridization instead of the RNase free paraffin sections, fixed in 4% paraformaldehyde and prepared using RNase protection procedures. RESULTS: Most of the paraffin sections, fixed in 10% neutral formalin solution in fresh condition, showed relatively good reaction of RNA in situ hybridization, although the necrotic tissue and autopsy specimens showed poor reaction of RNA in situ hybridization. A refixation procedure using a 4% paraformaldehyde solution was evaluated for optimal expression of mRNA in the paraffin sections. CONCLUSION: The treatment of 4% paraformaldehyde before the treatment of proteinase K showed better in situ hybridization than did the treatment of 4% paraformaldehyde after the treatment of proteinase K. Also a new Polymerase Chain Reaction (PCR)-based method of RNA probe production showed consistently good results.
Key Words: RNA; in situ hybridization; in situ hybridization; Polymerase Chain Reaction
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