An improved and combined reverse transcription-polymerase chain reaction assay for reliable detection of metastatic melanoma cells in peripheral blood.

The application of reverse transcription-polymerase chain reaction (RT-PCR) for detection of metastatic melanoma cells in peripheral blood has been used by a number of different scientific groups with both encouraging and discouraging results. We describe an improved, combined RT-PCR assay for tyrosinase mRNA, which can detect a single, viable melanoma cell in 10-15 ml of peripheral blood. The assay has been found to be highly sensitive, reproducible, cost-effective and clinically applicable. Furthermore, we developed a simple but effective procedure which prevents carry-over contamination. We present evidence indicating that the chance of obtaining normal melanocyte contamination with the needle prick during blood sampling is only 2% among all skin categories. It was found that the phenomenon of illegitimate transcription does not contribute to sporadic false positives. We conclude that the assay, as described here, is of clinical relevance and is a potentially powerful tool for studying various aspects of melanoma biology.