Improved Technique of Digoxigenin Labeled RNA in situ Hybridization.
- Author:
Suk Keun LEE
1
;
Yeon Sook KIM
;
In Sun SONG
;
Sang Shin LEE
;
Young Jun LEE
;
Woo Ho KIM
;
Je Geun CHI
Author Information
1. Department of Oral Pathology, Kangnung National University Dental College, Kangnung 210-711, Korea. sklee@knusun.kangnung.ac.kr
- Publication Type:Original Article
- Keywords:
RNA;
in situ hybridization;
in situ hybridization;
Polymerase Chain Reaction
- MeSH:
Autopsy;
Digoxigenin*;
Endopeptidase K;
Formaldehyde;
In Situ Hybridization*;
Paraffin;
Pathology;
Polymerase Chain Reaction;
Ribonucleases;
RNA Probes;
RNA*;
RNA, Messenger
- From:Korean Journal of Pathology
2001;35(2):98-110
- CountryRepublic of Korea
- Language:Korean
-
Abstract:
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.
