DETECTION OF TYROSINASE MESSENGER-RNA IN MELANOMA BY REVERSE TRANSCRIPTION-PCR AND ELECTROCHEMILUMINESCENCE

Increased sensitivity and improved quantitation of analytical tests us ed in biotechnology and clinical chemistry are goals of many laborator ies. We have used tyrosinase primers to specifically amplify by RT-PCR the tyrosinase mRNA expressed by the M12 melanoma cell Line in a back ground of mRNA from breast cancer cells. An electrochemiluminescence d etection procedure was used as a readout system for this study. Biotin ylated post-PCR cDNA samples were hybridized to a tris(2,2'-bipyridine )ruthenium(II) (TBR) chelate-labeled oligonucleotide probe, and the hy brid was subsequently captured by streptavidin-coated Dynabeads(R). Wh en either the QPCR System 5000 or the Origen 1 Analyzer System were us ed, the luminescence emitted by the TBR-chelate of the captured specif ic post-PCR product was assessed. Tyrosinase-specific mRNA isolated fr om similar to 1-10 melanoma cells in a background of 10(7) cells could be detected. We improved the sensitivity and logistics of the assay t hrough the use of rTth for reverse transcription and amplification Tyr osinase mRNA was detected in blood from 7 of 16 melanoma patients, whe reas none of the 5 healthy donor bloods were positive (P = 0.01; Wilco xon test).