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American Journal of Pathology, Vol 141, 579-589, Copyright © 1992 by American Society for Investigative Pathology

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Nonradioactive in situ hybridization using digoxigenin-labeled oligonucleotides. Applications to musculoskeletal tissues

ID Crabb, SS Hughes, DG Hicks, JE Puzas, GJ Tsao and RN Rosier
Department of Orthopaedics, University of Rochester, New York 14642.

We have optimized a technique for in situ localization of specific mRNAs using digoxigenin-11-dUTP-labeled oligonucleotide probes. DNA probes were synthesized for type I and type II collagen as well as transforming growth factor-beta 1 and 2 (TGF beta 1 and TGF beta 2). Control experiments, such as competitive inhibition, nonsense sequence hybridization, and RNAse digestion all indicated that the technique was highly sensitive and specific. In sections of growth plate, type II collagen mRNA was predominantly expressed in the lower proliferative and upper hypertrophic zone, whereas chondrocytes in articular cartilage stained equally. These techniques then were applied to sections cut from archival pathology specimens of musculoskeletal tissues. Primitive chondrocytes in a chondrosarcoma expressed type I and type II collagen mRNA, but did not stain with the nonsense probe. Sections from an osteosarcoma, an aneurysmal bone cyst, and a neurofibroma also were investigated. The ability to use chemically synthesized oligonucleotide probes, the high resolution, and the short development times possible with this in situ procedure makes this technique appealing for applied research into the gene expression of normal and pathologic cellular events.

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