Abstract
In situ polymerase chain reaction (PCR) is a recently developed technique whereby DNA (or cDNA) is enzymically amplified within cells or tissue sections, enabling the localization of specific low copy number sequences within a heterogeneous cell population. In situ reverse transcriptase PCR (RT-PCR) is an adaptation of the technique in which mRNA sequences are reverse transcribed to give a DNA copy (i.e., cDNA), which is subsequently amplified by PCR. The technique provides significantly enhanced sensitivity of detection over conventional mRNA in situ hybridization (ISH) as successful amplification can be achieved from a single mRNA target sequence. Amplification products are detected directly by incorporation of label during thermal cycling, or indirectly using a labeled probe and ISH (1-9). Figure 1 shows the basic principal of the technique.
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References
Chen, R. H. and Fuggle, S. V. (1997) In situ PCR amplification of intracellular mRNA. Methods Molec. Biol. 71, 123–132.
Long, A. A. and Komminoth, P. (1997) In situ PCR. An overview. Methods Molec. Biol. 71, 141–161.
Nuovo, G. J. (1994) In situ detection of PCR-amplified DNA and cDNA: a review. J. Histotechnol. 17, 235–246.
Teo, I. A. and Shaunak, S (1995). Polymerase chain reaction in situ: an appraisal of an emerging technique. Histochem. J. 27, 647–659.
Nuovo, G. J. (1992) PCR in situ Hybridization: Protocols and Applications. Raven, NewYork.
Lewis, F. (1996) An Approach to in situ PCR. PE Applied Biosystems, CA.
MartÍnez, A., Miller, M. J., Quinn, K., Unsworth, E. J., Ebina, M., and Cuttitta, F. (1995) Non-radioactive localisation of nucleic acids by direct in situ PCR and in situ RT-PCR in paraffin-embedded sections. J. Histochem. Cytochem. 43, 739–747.
Embelton, 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 with single cells. Nucleic Acids Res. 20, 3831–3837.
Waller, H. A. and Savage, A. K. (1994) Analysis of gene transcription in situ: methodological con-siderations and applications in pathology. J. Histotechnol. 17, 203–218.
Doyle, K., ed., (1996) Protocols and Applications Guide. Promega Corporation, Madison, WI.
Sambrook, J., Fritsch, E. F., and Maniatis, T. (1989) Molecular Cloning: A Laboratory Manual. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York.
Valentino, K. L., Eberwine, J. H., and Barchas, J. D., eds. (1987) In situ Hybridisation: Applications to Neurobiology. Oxford University Press, Oxford, England.
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Anne Waller, H., Kay Savage, A. (2000). mRNA Detection by In Situ RT-PCR. In: Bartlett, J.M.S. (eds) Ovarian Cancer. Methods in Molecular Medicine™, vol 39. Humana Press. https://doi.org/10.1385/1-59259-071-3:417
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DOI: https://doi.org/10.1385/1-59259-071-3:417
Publisher Name: Humana Press
Print ISBN: 978-0-89603-583-6
Online ISBN: 978-1-59259-071-1
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