- Improved Technique of Digoxigenin Labeled RNA in situ Hybridization.
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Suk Keun Lee, Yeon Sook Kim, In Sun Song, Sang Shin Lee, Young Jun Lee, Woo Ho Kim, Je Geun Chi
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Korean J Pathol. 2001;35(2):98-110.
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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.
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.
- Molecular Cloning of Novel Genes Related to the Craniofacial Development of Human Embryo.
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Young Jun Lee, Tak Soo Go, Hyung Wook Han, Sang Shin Lee, Eun Cheol Kim, Yeon Sook Kim, Suk Keun Lee, Je G Chi
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Korean J Pathol. 2000;34(12):961-971.
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Abstract
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- In order to obtain novel genes for craniofacial development of human, molecular cloning and sequencing were performed and followed by in situ hybridization in tissue sections. Subtracted cDNA library of craniofacial tissue from 8 weeks old human embryo was made by the subtraction with cDNA of RHEK cells.
A total of 231 clones were obtained and their partial sequence data disclosed that 214 clones were nonredundant in Genebank search. We have done in situ hybridization screening on the craniofacial sections of a 10 weeks old human fetus, and found significant positive reaction in 30 clones. Depending on the cell type of similar developmental origin, the positive reactions could be divided into four groups: first group showed an intense positive reaction in neural tube, ganglion, and a part of peripheral nerve tissue, second group relatively diffuse positive reaction in neural tube, cartilage, epithelium, and muscle, third group localized positive reaction in nerve, and muscle, and fourth group positive reaction in almost all kinds of cells of craniofacial tissues. Although every clone showed different expression patterns in the craniofacial development, some of them showed intense mRNA expressions in the characteristic cell type.
Because this study also aimed to test a screening methods to find out novel genes related to craniofacial development by the subtracted cDNA library and in situ hybridization, the intense positive reaction of a certain clone by in situ hybridization may indicate its role in the developmental processes. We presumed that 30 clones selected in this study are possibly important new genes for the development of human craniofacial structure.
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