Quantitative nested real-time RT-PCR specific for tyrosinase transcripts to quantitate minimal residual disease

Abstract

Background: Quantification of tumor markers expressed by occult-circulating tumor cells may be of prognostic value in a variety of neoplasms and disease stages. Here, we report on a quantitative nested real-time polymerase chain reaction (PCR) assay to quantify rare transcripts using the Light Cycler system. Tyrosinase mRNA, only expressed by melanocytes and melanoma cells, was used as the model tumor marker. Methods: For the establishment and sensitivity testing of this novel real-time PCR assay, 10 ml EDTA blood from healthy volunteers was spiked with 10 1–10 5 MKR cells (the melanoma cell line). Following RNA extraction and cDNA synthesis, nested and single round PCRs were performed. Subsequently, 35 blood samples of 22 melanoma patients were analyzed. Results: Nested PCRs provided quantitative data with a quantitative range of five orders of magnitude of tyrosinase mRNA equivalent to 1–10 4 MKR cells/ml of blood. Nested PCR with 35 pre-amplification cycles displayed the quantitative data for nine tyrosinase transcript-positive samples out of 35 blood samples, whereas nested PCR with 20 pre-amplification cycles or single round PCR were less sensitive. Conclusions: These experiments indicate that nested PCR, which is much more sensitive than single round PCR, is a useful tool even for the quantification of rare transcripts—a result with important implications for the study of minimal residual disease (MRD) in melanoma as well as in other malignancies.