Abstract
Gene expression in bone can be assessed by several techniques such as reverse transcription polymerase chain reaction (RT-PCR), differential display PCR, subtractive hybridization, and microchip arrays. The problem with all of these techniques is that they do not allow cellular localization of the genes under investigation. In situ hybridization (ISH) circumvents this problem. It is possible to localize precisely sites of gene expression using ISH in intact cells and tissue sections, allowing one to build up a more complete picture of the processes occurring in the disease under investigation.
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References
Komminoth, P. and Long, A. A. (1993) In-situ polymerase chain reaction. An overview of methods, applications and limitations of a new molecular technique. Virchows Arch. B 64, 67–73.
Haase, A. T., Retzel, E. F., and Staskus, K. A. (1990) Amplification and detection of Lentiviral DNA inside cells. Proc. Natl. Acad. Sci. USA 87, 4971–4975.
Nuovo, G. J., Gallery, F., MacConnell, P., Becker, J., and Bloch, W. (1991) An improved technique for in situ detection of DNA after polymerase chain reaction ampification. Am. J. of Pathol. 139, 1239–1244.
Bagasra, O., Hauptman, S. P., Lishner, H. W., Sachs, M., and Pomerantz, R. J. (1992) Detection of human immunodeficiency virus type 1 provirus in mononuclear cells by in situ polymerase chain reaction. N. Engl. J. Med. 326, 1385–1391.
Zehbe, I., Hacker, G. W., Rylabder, E., Sallstrom, J., and Wilander, E. (1992) Detection of single HPV copies in SiHa cells by in situ polymerase chain reaction combined with immunoperoxidase and immunogold-silver staining techniques. Anticancer Res. 12, 2165–2168.
Komminoth, P., Long, A., Ray, R., and Wolfe, H. (1992) In situ polymerase chain reaction detection of viral DNA, single copy genes and gene rearrangements in cell suspensions and cytospins. Diagn. Mol. Pathol. 1, 85–97.
Embleton, M. J., Gorochov, G., Jones, P. T., and Winter, G. (1992) In-cell PCR from mRNA: amplifying and linking the rearranged immunoglobulin heavy and light chain V-genes within single cells. Nucl. Acids Res. 20, 3831–3837.
Heniford, B. W., Shum-Siu, A., Leonberger, M., and Hendler, F. J. (1993) Variation in cellular EGF receptor mRNA expression demonstrated by in situ reverse transcriptase polymerase chain reaction. Nucl. Acids Res. 21, 3159–3166.
Chen, R. H. and Fuggle, S. V. (1993) In situ cDNA polymerase chain reaction. A novel technique for detecting mRNA expression. Am. J. Pathol. 143, 1527–1534.
Nuovo, G. J. (1994) Reverse transcriptase in situ PCR, in PCR In Situ Hybridization. Protocols and Applications, 2nd edit. Raven Press, New York, pp. 247–306.
Patel, V. G., Shum-Siu, A., Heniford, B. W., Wieman, T. J., and Hendler, F. J. (1994) Detection of epidermal growth factor receptor mRNA in tissue sections from biopsy specimens using in situ polymerase chain reaction. Am. J. Pathol. 144, 7–14.
Martinez, A., Miller, M. J., Quinn, K., Unsworth, E. J., Ebina, M., and Cuttitta, F. (1995) Non-radioactive localization of nucleic acids by direct in situ PCR and in situ RT-PCR in paraffin-embedded sections. J. Histochem. Cytochem. 43, 739–747.
Martinez, A., Miller, M. J., Unsworth, E. J., Stegfried, J. M., and Cuttitta, F. (1995) Expression of adrenomedullin in normal human lung and in pulmonary tumours. Endocrinology 136, 4099–4105.
Mee, A. P., Davenport, L. K., Hoyland, J. A., Davies, M., and Mawer, E. B. (1996) Novel and sensitive detection systems for the vitamin D receptor—in situ-reverse transcriptase-polymerase chain reaction and immunogold cytochemistry. J. Mol. Endocrinol. 16, 183–195.
Mee, A. P., Hoyland, J. A., Braidman, I. P., Freemont, A. J., Davies, M., and Mawer, E. B. (1996) Demonstration of vitamin D receptor transcripts in actively resorbing osteoclasts in bone sections. Bone 18, 295–299.
Mee, A. P., Denton, J., Hoyland, J. A., Davies, M., and Mawer, E. B. (1997) Quantification of vitamin D receptor mRNA in tissue sections demonstrates the relative limitations of in situ-reverse transcriptase-polymerase chain reaction. J. Pathol. 182, 22–28.
Hoyland, J. A., Mee, A. P., Baird, P., Braidman, I. P., Mawer, E. B., and Freemont, A. J. (1997) Demonstration of oestrogen receptor mRNA in bone using in situ-reverse transcriptase-polymerase chain reaction. Bone 20, 87–92.
Mee, A. P., Dixon, J. A., Hoyland, J. A., Davies, M., Selby, P. L., and Mawer, E. B. (1998) Detection of canine distemper virus in 100% of Paget’s disease samples by in situ-reverse transcriptase-polymerase chain reaction. Bone 23, 171–175.
Long, A. A., Komminoth, P., Lee, E., and Wolfe, H. J. (1993) Comparison of indirect and direct in situ polymerase chain reaction in cell preparations and tissue sections. Histochemistry 99, 151–162.
Myers, T. W. and Gelfand, D. H. (1991) Reverse transcription and DNA amplification by a Thermus thermophilus DNA polymerase. Biochemistry 30, 7661.
Walsh, L., Freemont, A. J., and Hoyland, J. A. (1993) The effect of tissue decalcification on mRNA retention within bone for in situ hybridization studies. Int. J. Exp. Pathol. 74, 237–241.
Rentrop, M., Knapp, B., Winter, H., and Schweizer, J. (1986) Aminoalkylsilane-treated slides as support for in situ hybridization of keratin cDNAs to frozen sections under varying fixation and pretreatment conditions. Histochem. J. 18, 271–276.
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© 2003 Humana Press Inc., Totowa, NJ
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Mee, A.P., Hoyland, J.A. (2003). In Situ Hybridization and In Situ Reverse Transcription Polymerase Chain Reaction in Human Bone Sections. In: Helfrich, M.H., Ralston, S.H. (eds) Bone Research Protocols. Methods in Molecular Medicine, vol 80. Humana Press. https://doi.org/10.1385/1-59259-366-6:201
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DOI: https://doi.org/10.1385/1-59259-366-6:201
Publisher Name: Humana Press
Print ISBN: 978-1-58829-044-1
Online ISBN: 978-1-59259-366-8
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