[Frontiers in Bioscience 1, c4-15, November 1, 1996]
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CAVEAT LECTOR



THE FOUNDATION OF SUCCESSFUL RT IN SITU PCR

Gerard J. Nuovo

Director, MGN Medical Research Laboratories, Setauket, New York 11733, USA

Received: 07/25/96; Accepted: 10/02/96; On-line 11/01/96

2. INTRODUCTORY STATEMENT

The field involving the in situ detection of PCR-amplified DNA and cDNA has advanced considerably in the last 5 years. A consequence of this rapid movement has been a proliferation of laboratory protocols and procedures and their accompanying "camps" of supporters. These differing approaches have led to many ideas that, over time and in the light of new data, have, in my opinion, been shown to be incorrect. For example, one can use full length probes for PCR in situ hybridization, with their much greater signal to background ratio, as they do not per se lead to background due to the apparent lack of detectable primer oligomerization inside the cell (1). Also, a high stringency wash after RT in situ PCR can eliminate the background that may result from nonspecific binding of labeled primer oligomers that form in the amplifying solution during the procedure (1). There are several other misconceptions and either incorrect or partly correct statements regarding in situ PCR that may be hindering the advancement of the field. In particular:

  • RT in situ PCR (that is, direct incorporation of the reporter nucleotide) invariably leads to false positive results;
  • migration of the amplicon during RT in situ PCR may lead to false positive results when the amplicon sticks to a cell that does not have the target (2-16). Included in the reference listing (2-16) are the protocols of many different laboratories who have reported data using in situ PCR. This list is presented as alternative techniques for doing in situ PCR as well as discussions of potential problems with the technique.

In understanding the keys to successful RT in situ PCR, it is important to appreciate some differences between doing solution phase PCR in a 50 µl volume in a GeneAmp tube and PCR in a cell of 5 microns with less than 1,000th of the volume. First, there is the marked difference in the surface to volume ratio. In a 0.5 ml tube, the ratio of the surface area to volume is about 1:2. With RT in situ PCR, assuming that one uses a 10 mm coverslip and a volume of 10 µl, the surface to volume ratio is over 20 times greater. This will alter the cycling parameters one uses for RT in situ PCR, especially the time and temperature of denaturation (17). The second difference is that the amplifying solution in solution phase PCR is mostly water, and the test sample provides relatively scanty amounts of DNA and even smaller amounts of proteins. With RT in situ PCR, the nucleus of a cell provides a relatively dense matrix of DNA, RNA and proteins which, if fixed in formalin, will be extensively cross linked to form a complex, 3-dimensional labyrinth. The cytoplasm of the cell also consists of a complex 3-dimensional matrix that is composed of its "skeleton", made primarily of the intermediate filaments, and a wide variety of other proteins and RNA. The relatively dense and complex protein-nucleic acid matrix found in the cell cytoplasm can be exploited to serve as an "anchor" for the amplicon to prevent its migration out of the cell. The relatively concentrated collection of proteins and nucleic acids in a fixed cell are the basis of some fundamental differences in the various DNA synthesis pathways that can be operative during in situ PCR versus solution phase PCR.

The first part of this review will focus on the key variables for successful RT in situ PCR. These key steps include sample fixation, protease digestion and DNase digestion. This section will be followed by a protocol for RT in situ PCR using the one step rTth system. Next detection of matrix metalloprotease mRNAs in cervical cancer cells will be used as a model of the RT in situ PCR technique, focusing on the interpretation of the positive and negative controls and the importance of the specific localization of the signal, that is, nuclear versus cytoplasmic.

Before beginning the review, the following terms should be defined:

  • in situ PCR - direct incorporation of the reporter nucleotide into the PCR product as it is being synthesized within the cell.
  • RT in situ PCR - detection of PCR-amplified cDNA via direct incorporation of the reporter nucleotide.
  • PCR in situ hybridization - detection of the PCR product with a labeled probe after it is synthesized within the cell using a hybridization step.

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