Application of quantitative RT-PCR using "TaqMan" technology to evaluate the expression of CK 18 mRNA in various cell lines

Reverse transcriptase polymerase chain reaction (RT-PCR) is often used for sensitive detection of micrometastasis in peripheral blood, lymph nodes and bone marrow. While the utility of this method has been documented, it also has limitations in the detection of micrometastasis. The mRNA of target ge nes can be detected in healthy donors or in samples used for negative contr ol, therefore the non-quantitativeness of conventional RT-PCR has been call ed into question. We analyzed the expression level of cytokeratin (CK) 18 mRNA in established esophageal and gastrointestinal carcinoma cell lines and non-epithelial ce lls, using quantitative RT-PCR, based on real time 'TaqMan TM' technology. CK 18 mRNA is more highly expressed in carcinoma cells than in non-epitheli al cells. However, the expression level in non-epithelial cells was easily detected using conventional RT-PCR and agarose gel electrophoresis. In an a nalysis of CK 18 mRNA expression in peripheral venous blood in 13 healthy v olunteers, we found that CK 18 mRNA was much less expressed than in cancer cell lines. However, the expression in all samples was at a level which was also detected using conventional RT-PCR. It would thus seem that not only qualitative, but also quantitative analysis, of the target mRNA is importan t to detect micrometastasis. Quantitative RT-PCR methods will make comparis ons of the possible differences in expression levels of the target gene. Fo r clinical applications, much further study is needed.