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KMID : 0357920010350020098
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Korean Journal of Pathology
2001 Volume.35 No. 2 p.98 ~ p.110
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Improved Technique of Digoxigenin Labeled RNA in situ Hybridization
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Lee Suk-Keun
Kim Yeon-Sook
Song In-Sun
Lee Sang-Shin
Lee Young-Joon
Kim Woo-Ho
Chi Je-Geun
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Abstract
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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.
Conclusions: 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.
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KEYWORD
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RNA, in situ hybridization, Digoxigenin, Polymerase Chain Reaction
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