Korean J Pathol.  2001 Apr;35(2):98-110.

Improved Technique of Digoxigenin Labeled RNA in situ Hybridization

• Lee SK
• Kim YS
• Song IS
• Lee SS
• Lee YJ
• Kim WH
• Chi JG

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.

Keyword

RNA; in situ hybridization; in situ hybridization; Polymerase Chain Reaction

MeSH Terms

Autopsy
Digoxigenin*
Endopeptidase K
Formaldehyde
In Situ Hybridization*
Paraffin
Pathology
Polymerase Chain Reaction
Ribonucleases
RNA Probes
RNA*
RNA, Messenger
Digoxigenin
Endopeptidase K
Formaldehyde
Paraffin
RNA
RNA Probes
RNA, Messenger
Ribonucleases