Immunobead RT-PCR versus regular RT-PCR amplification of CEA mRNA in peripheral blood

Purpose: The reverse transcription polymerase chain reaction (RT-PCR) ampli fication of tumor-specific mRNA has been used for the detection of cancer c ells in peripheral blood. More recently, an immunomagnetic isolation and re verse transcription polymerase chain reaction (immunobead RT-PCR) was devel oped which has reportedly significant advantages over the previous RT-PCR a nalysis. In our study, we compared these two methods using a model set of p eripheral blood containing tumor cells under standardized conditions. Mater ial and methods: In order to compare the false positive rate, normal periph eral blood samples from five volunteers were analyzed by both methods. A mo del set of peripheral blood containing tumor cells was established by addin g SNUC4 human colon cancer cells to peripheral blood collected from normal volunteers not showing any nonspecific bands upon electrophoresis of the PC R products. RT-PCR amplification of carcinoembryonic antigen (CEA) mRNA was done with total RNA and mRNA prepared from this model sample. In immunobea d RT-PCR analysis, mRNA was prepared from the cells isolated with anti-CEA antibody-coated magnetic beads or anti-Ber-EP4 antibody-coated magnetic bea ds before the RT-PCR analysis. Result: The immunobead RT-PCR yielded no non -specific band, while the regular RT-PCR using total RNA did show non-speci fic band formation in all five samples. When mRNA rather than total RNA was used, nonspecific bands were formed in three of the five samples. Immunobe ad RT-PCR allowed the detection of 10(1) tumor cells in 1 ml of peripheral blood. The regular RT-PCR analysis had a detection limit of 10(2) tumor cel ls in 1 ml of peripheral blood. Conclusion: The immunobead RT-PCR proved to be more sensitive and specific than the regular RT-PCR at least in our mod el system.