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Special PCR Applications In Situ PCR
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In
Situ
Polymerase Chain Reaction: Dr. med. Paul Komminoth Note: The article presented below originally appeared in the 1997 ”Boehringer Mannheim PCR Bibliography on the Occasion of the PCR Award for Young Scientists” (BM publication no. 1675044).
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Introduction In situ hybridization (ISH) has
proven to be a very important molecular tool in diagnostic and research
and has significantly advanced the study of gene structure and
expression at the level of individual cells. However, the usefulness of
ISH is occasionally limited by a detection sensitivity of about 20
copies of mRNA per cell, an obstacle that may now be overcome by
exciting new technologies. In recent years, strategies to improve threshold levels for detection in ISH studies have included protocols which either
amplify the detection signals e.g. using several antibody steps
or increase the absolute amount of hybridized probes by using e.g. cocktails of oligonucleotide or multiple cRNA probes. A third
strategy employing a PCR based amplification of the target nucleic
acid sequences prior to ISH was developed by several laboratories
independently in the late 1980’s. This approach has variously been
termed ”in situ PCR”, ”PCR in situ”, ”PCR in
situ hybridization”, ”in-cell PCR”, ”PCR-driven ISH” or
”cycling PAINS”. More recently, an intracellular reverse
transcription (RT) step to generate complementary DNA from mRNA
templates prior to in situ PCR has been used for the detection of low
copy mRNA sequences. This modification of in situ PCR has been
termed ”in situ RT-PCR” or less precisely ”RT in situ PCR” or ”in situ
cDNA PCR”. Principles
and Methods Experimental protocols for successful in situ PCR have been developed by several groups and share important key steps. These include fixation and permeabilization during sample preparation, a mechanism for thermal cycling cellular material in solution or on glass slides and a means to detect the amplificants (6, 8). Variables at each of these steps that likely account for some of the reported |
discrepancies in both results and interpretation have been
identified and certain principles of optimization are now emerging (5,
6, 8).
Prior to in situ PCR, cells or tissue samples are fixed and permeabilized to preserve morphology and permit access of the
PCR reagents to the intracellular sequences to be amplified. PCR
amplification of target sequences is next performed either in intact cells
held in suspension (Fig. 90) in micro-Eppendorf tubes or directly in
cytocentrifuge preparations or tissue sections on glass slides
(Fig. 91). In the former approach, fixed cells suspended in the PCR
reaction mixture are thermal cycled in micro-Eppendorf tubes using
conventional block cyclers. After PCR the cells are cytocentrifugated
onto glass slides with visualization of intracellular PCR products by
ISH or immunohistochemistry. In situ PCR on glass slides is
performed by overlaying the samples with the PCR mixture under a
coverslip which is then sealed with nail polish, rubber cement or
mineral oil to prevent evaporation of the reaction mixture. Thermal
cycling is achieved by placing the glass slides either directly on top of the heating block of a conventional or
specially designed thermal cycler or by using thermal cycling ovens.
Detection of intracellular PCR-products is achieved by one of two
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