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

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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 HCl 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.

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