A Sensitive Method of Non-Radioactive In Situ Hybridization for mRNA Localization within Human Renal Biopsy Specimens: Use of Digoxigenin Labeled Oligonucleotides

Masanobu Miyazaki, Masayuki Endoh, Yasuo Nomoto, Hideto Sakai, David J. Nikolic-Paterson, Robert C. Atkins, Takehiko Koji

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23 Citations (Scopus)

Abstract

A sensitive method of non-radioactive in situ hybridization using digoxigenin-labeled oligonucleotides is described for the detection of mRNA within human renal biopsy specimens. Although non-radioactive in situ hybridization typically has the drawback of low sensitivity, we increased the sensitivity of this method, providing a practical alternative to the use of radiolabelled probes. The four main points are: 1) assessment of the efficiency of labeling, 2) optimization of the probe concentration for hybridization, 3) requirement of deproteinization of tissues with HC1 and proteinase K, and 4) the use of a four-layer immunoperoxidase staining system. This technique was found to clearly localize individual mRNA positive cells within cryostat tissue sections. A variety of controls including sense probes, excess unlabeled anti-sense probes, and RNase-treatment demonstrated the specificity of the technique. This improved method is a powerful technique for detecting mRNA within human renal tissue and will be most useful in the study of gene expression in the pathogenesis of renal diseases. (Internal Medicine 33: 87-91, 1994).

Original languageEnglish
Pages (from-to)87-91
Number of pages5
JournalInternal Medicine
Volume33
Issue number2
DOIs
Publication statusPublished - 1 Jan 1994
Externally publishedYes

Keywords

  • digoxigenin
  • IL-6
  • in situ hybridization
  • renal biopsy

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