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. 1992 Sep;141(3):579–589.

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

I D Crabb 1, S S Hughes 1, D G Hicks 1, J E Puzas 1, G J Tsao 1, R N Rosier 1
PMCID: PMC1886689  PMID: 1519665

Abstract

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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Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Crabb I. D., O'Keefe R. J., Puzas J. E., Rosier R. N. Differential effects of parathyroid hormone on chick growth plate and articular chondrocytes. Calcif Tissue Int. 1992 Jan;50(1):61–66. doi: 10.1007/BF00297299. [DOI] [PubMed] [Google Scholar]
  2. Crabb I. D., O'Keefe R. J., Puzas J. E., Rosier R. N. Synergistic effect of transforming growth factor beta and fibroblast growth factor on DNA synthesis in chick growth plate chondrocytes. J Bone Miner Res. 1990 Nov;5(11):1105–1112. doi: 10.1002/jbmr.5650051103. [DOI] [PubMed] [Google Scholar]
  3. Devlin C. J., Brickell P. M., Taylor E. R., Hornbruch A., Craig R. K., Wolpert L. In situ hybridization reveals differential spatial distribution of mRNAs for type I and type II collagen in the chick limb bud. Development. 1988 May;103(1):111–118. doi: 10.1242/dev.103.1.111. [DOI] [PubMed] [Google Scholar]
  4. Heiles H. B., Genersch E., Kessler C., Neumann R., Eggers H. J. In situ hybridization with digoxigenin-labeled DNA of human papillomaviruses (HPV 16/18) in HeLa and SiHa cells. Biotechniques. 1988 Nov-Dec;6(10):978–981. [PubMed] [Google Scholar]
  5. Heino P., Hukkanen V., Arstila P. Detection of human papilloma virus (HPV) DNA in genital biopsy specimens by in situ hybridization with digoxigenin-labeled probes. J Virol Methods. 1989 Dec;26(3):331–337. doi: 10.1016/0166-0934(89)90115-8. [DOI] [PubMed] [Google Scholar]
  6. Herrington C. S., Burns J., Graham A. K., Bhatt B., McGee J. O. Interphase cytogenetics using biotin and digoxigenin labelled probes II: Simultaneous differential detection of human and papilloma virus nucleic acids in individual nuclei. J Clin Pathol. 1989 Jun;42(6):601–606. doi: 10.1136/jcp.42.6.601. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Herrington C. S., Burns J., Graham A. K., Evans M., McGee J. O. Interphase cytogenetics using biotin and digoxigenin labelled probes I: relative sensitivity of both reporter molecules for detection of HPV16 in CaSki cells. J Clin Pathol. 1989 Jun;42(6):592–600. doi: 10.1136/jcp.42.6.592. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Leboy P. S., Shapiro I. M., Uschmann B. D., Oshima O., Lin D. Gene expression in mineralizing chick epiphyseal cartilage. J Biol Chem. 1988 Jun 15;263(17):8515–8520. [PubMed] [Google Scholar]
  9. Mallein-Gerin F., Kosher R. A., Upholt W. B., Tanzer M. L. Temporal and spatial analysis of cartilage proteoglycan core protein gene expression during limb development by in situ hybridization. Dev Biol. 1988 Apr;126(2):337–345. doi: 10.1016/0012-1606(88)90144-3. [DOI] [PubMed] [Google Scholar]
  10. McDonald S. A., Tuan R. S. Expression of collagen type transcripts in chick embryonic bone detected by in situ cDNA-mRNA hybridization. Dev Biol. 1989 May;133(1):221–234. doi: 10.1016/0012-1606(89)90313-8. [DOI] [PubMed] [Google Scholar]
  11. O'Keefe R. J., Crabb I. D., Puzas J. E., Rosier R. N. Countercurrent centrifugal elutriation. High-resolution method for the separation of growth-plate chondrocytes. J Bone Joint Surg Am. 1989 Apr;71(4):607–620. [PubMed] [Google Scholar]
  12. Oshima O., Leboy P. S., McDonald S. A., Tuan R. S., Shapiro I. M. Developmental expression of genes in chick growth cartilage detected by in situ hybridization. Calcif Tissue Int. 1989 Sep;45(3):182–192. doi: 10.1007/BF02556062. [DOI] [PubMed] [Google Scholar]
  13. Sandberg M., Vuorio E. Localization of types I, II, and III collagen mRNAs in developing human skeletal tissues by in situ hybridization. J Cell Biol. 1987 Apr;104(4):1077–1084. doi: 10.1083/jcb.104.4.1077. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Swalla B. J., Upholt W. B., Solursh M. Analysis of type II collagen RNA localization in chick wing buds by in situ hybridization. Dev Biol. 1988 Jan;125(1):51–58. doi: 10.1016/0012-1606(88)90057-7. [DOI] [PubMed] [Google Scholar]
  15. Wolfson B., Manning R. W., Davis L. G., Arentzen R., Baldino F., Jr Co-localization of corticotropin releasing factor and vasopressin mRNA in neurones after adrenalectomy. Nature. 1985 May 2;315(6014):59–61. doi: 10.1038/315059a0. [DOI] [PubMed] [Google Scholar]
  16. Zischler H., Nanda I., Schäfer R., Schmid M., Epplen J. T. Digoxigenated oligonucleotide probes specific for simple repeats in DNA fingerprinting and hybridization in situ. Hum Genet. 1989 Jun;82(3):227–233. doi: 10.1007/BF00291160. [DOI] [PubMed] [Google Scholar]

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