In situ reverse transcription-polymerase chain reaction. Applications for light and electron microscopy.
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.
Morel
G
G
CNRS-UMR 5578, Université Claude-Bernard-Lyon 1, Villeurbanne, France.
Berger
M
M
Ronsin
B
B
Recher
S
S
Ricard-Blum
S
S
Mertani
H C
HC
Lobie
P E
PE
D000818:Animals; D004247:DNA / Q000032:analysis* / Q000235:genetics; D017931:DNA Primers; D018076:DNA, Complementary / Q000032:analysis; D004279:DNA, Viral / Q000032:analysis; D004058:Diffusion; D013006:Growth Hormone / Q000235:genetics; D017403:In Situ Hybridization / Q000379:methods*; D015636:Magnesium Chloride / Q000494:pharmacology; D008297:Male; D051379:Mice; D008853:Microscopy / Q000379:methods*; D008854:Microscopy, Electron / Q000379:methods*; D010903:Pituitary Gland, Anterior / Q000737:chemistry / Q000648:ultrastructure; D016133:Polymerase Chain Reaction / Q000379:methods*; D012333:RNA, Messenger / Q000032:analysis; D012367:RNA, Viral / Q000032:analysis; D051381:Rats; D012680:Sensitivity and Specificity; D013048:Specimen Handling; D013696:Temperature; D013997:Time Factors; D016707:Tissue Fixation; D014780:Viruses / Q000302:isolation & purification
D017931:DNA Primers; D018076:DNA, Complementary; D004279:DNA, Viral; D012333:RNA, Messenger; D012367:RNA, Viral; D015636:Magnesium Chloride; D013006:Growth Hormone; D004247:DNA