In Situ PCR to Cells and to Wax Sections.

In situ hybridization and solution-phase PCR are suitable methods for DNA or RNA analysis. The first protocol for in situ hybridization was described almost 25 years ago (1). Since then, this technique has proven valuable to localize cellular DNA or mRNA. It has also been applied to detect viral DNA or RNA nucleic acid sequences in tissue sections or individual cells. The sensitivity of this procedure is, however, limited when small amounts of nucleic acids have to be detected (2-10). Solution-phase PCR, the most sensitive assay for detecting the presence of low-copy number RNA or DNA, has been frequently used over the past 10 years in many fields (for review, see 11). It permits the selective in vitro amplification of a specific region of nucleic acid by mimicking the phenomenon of in vivo DNA replication (12). It has been extensively used to amplify specific nucleic acid sequences for analysis of viral pathogens. It has successfully improved the detection of human cytomegalovirus (CMV) and human papillomaviruses (HPV) (13-19). This method is currently limited in application, since localizing amplified sequences inside cells or tissue sections is impossible. Recently, some authors have described a new procedure combining in situ hybridization and PCR, and allowing the detection of a low abundance of target sequences in cultured or cytospun cells and in frozen or wax-embedded tissue sections. This technique has been called in situ PCR, PCR in situ, PCR in situ hybridization, in cell PCR, and PCR driven in situ hybridization. Several protocols have been described so far, and the authors have stressed the importance of various fixatives, fixation time, and proteolytic digestion to preserve cellular morphology and to avoid leakage of the amplified products (20-38). Thus, substantial differences between the sophisticated procedures may affect the reproducibility of the results. The amplification of low-copy target DNA or even cDNA (after a reverse transcription step) can be achieved by different protocols for in situ PCR. Direct in situ PCR is mostly conducted with the use of biotin or digoxigenin-labeled nucleotides or primers, and the visualization of the PCR products is performed using an immunohistochemistry method. Indirect in situ PCR is performed with unlabeled nucleotides or primers, and the amplified products are detected by a classical in situ hybridization using specific probes. [ABSTRACT FROM AUTHOR]