Kvantitativna in situ hibridizacija povećane osjetljivosti u mekom, koštanom i zubnom tkivu pomoću RNA probi označenih digoksigeninom

Translated title of the contribution: Quantitative in situ hybridization with enhanced sensitivity in soft, bone and tooth tissue using digoxigenin tagged RNA probes

Jelica Gluhak-Heinrich, Wuchen Yang, Marie A. Harris, Stephen E. Harris

Research output: Contribution to journalArticle

6 Scopus citations

Abstract

Introduction: Quantitative non-radioactive in situ hybridization is a powerful technique for localizing the expression of mRNA transcripts. These methods enable mRNAs to be detected with great resolution on a single cell level. Up to date the most published non-radioactive protocols used thick cryostat sections in soft tissue localizing high abundant genes without quantification. Material and methods: We developed a non-radioactive in situ hybridization method using thin sections of demineralized bone paraffin embedded tissue with low abundant gene detection and in situ signal quantification. Our protocol is based on the optimal synthesis of digoxigenin labeled RNA probes to visualize very low abundant genes in soft, bone and tooth paraffin embedded tissues. Our new technique visualizes an in situ signal with amplification and enhanced color by developing alkaline phosphatase reaction. Results: The sensitivity is at the level of radioactivity and the resolution is at the level of a single cell, which is better than with radioactivity. Quantification of in situ hybridization signal, previously used with radioactive or fluorescent labeling, is now possible with alkaline phosphatase using our technique and ImageJ program. Conclusion: The presented examples show that the following method has better proven resolution and equivalent sensitivity to radioactive labeled methods in different tissues with quantification ability, thus indicating that this method can generally be used in research and clinical laboratories.

Translated title of the contributionQuantitative in situ hybridization with enhanced sensitivity in soft, bone and tooth tissue using digoxigenin tagged RNA probes
Original languageCroatian
Pages (from-to)59-80
Number of pages22
JournalBiochemia Medica
Volume18
Issue number1
StatePublished - Mar 19 2008

Keywords

  • Digoxigenin probes
  • Praffin embedded bone tissue
  • Quantitative in situ hybridization

ASJC Scopus subject areas

  • Clinical Biochemistry
  • Biochemistry, medical

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