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Title: In situ reverse transcription-polymerase chain reaction. Applications for light and electron microscopy
Authors: Morel, G.
Berger, M.
Ronsin, B.
Recher, S.
Ricard-Blum, S.
Mertani, H.C. 
Lobie, P.E. 
Keywords: Electron microscopy
Growth hormone receptor mRNA
In situ RT-PCR light microscopy
Virus amplification
Issue Date: Mar-1998
Citation: Morel, G., Berger, M., Ronsin, B., Recher, S., Ricard-Blum, S., Mertani, H.C., Lobie, P.E. (1998-03). In situ reverse transcription-polymerase chain reaction. Applications for light and electron microscopy. Biology of the Cell 90 (2) : 137-154. ScholarBank@NUS Repository.
Abstract: Since its discovery in 1986 by Mullis, the polymerase chain reaction (PCR) has been extensively developed by morphologists in order to overcome the main limitation of in situ hybridization, the lack of sensitivity. In situ PCR combines the extreme sensitivity of PCR with the cell-localizing ability of in situ hybridization. The amplification of DNA (PCR) or a cDNA (RT-PCR) in cell or tissue sections has been developed at light and electron microscopic levels. A successful PCR experiment requires the careful optimization of several parameters depending on the tissue (or of cell types), and a compromise must be found between the fixation time, pretreatments and a good preservation of the morphology Other crucial factors (primer design, concentration in MgCl2, annealing and elongation temperatures during the amplification steps) and their influence on the specificity and sensitivity of in situ PCR or RT-PCR are discussed. The necessity to run appropriate controls, especially to assess the lack of diffusion of the amplified products, is stressed. Current applications and future trends are also presented.
Source Title: Biology of the Cell
ISSN: 02484900
DOI: 10.1016/S0248-4900(98)80335-3
Appears in Collections:Staff Publications

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