Detection of tyrosinase mRNA in melanoma by reverse transcription-PCR and electrochemiluminescence.
Increased sensitivity and improved quantitation of analytical tests used in biotechnology and clinical chemistry are goals of many laboratories. We have used tyrosinase primers to specifically amplify by RT-PCR the tyrosinase mRNA expressed by the M12 melanoma cell line in a background of mRNA from breast cancer cells. An electrochemiluminescence detection procedure was used as a readout system for this study. Biotinylated post-PCR cDNA samples were hybridized to a tris(2,2'-bipyridine)ruthenium(II) (TBR) chelate-labeled oligonucleotide probe, and the hybrid was subsequently captured by streptavidin-coated Dynabeads. When either the QPCR System 5000 or the Origen 1 Analyzer System were used, the luminescence emitted by the TBR-chelate of the captured specific post-PCR product was assessed. Tyrosinase-specific mRNA isolated from approximately 1-10 melanoma cells in a background of 10(7) cells could be detected. We improved the sensitivity and logistics of the assay through the use of rTth for reverse transcription and amplification. Tyrosinase mRNA was detected in blood from 7 of 16 melanoma patients, whereas none of the 5 healthy donor bloods were positive (P = 0.01; Wilcoxon test).
O'Connell
C D
CD
Biotechnology Division, National Institute of Standards and Technology, Gaithersburg, MD 20899, USA. coc@nist.gov
Juhasz
A
A
Kuo
C
C
Reeder
D J
DJ
Hoon
D S
DS
D001943:Breast Neoplasms / Q000473:pathology; D004259:DNA-Directed DNA Polymerase; D004560:Electricity; D005260:Female; D006801:Humans; D008163:Luminescent Measurements; D008545:Melanoma / Q000097:blood / Q000201:enzymology* / Q000473:pathology; D014442:Monophenol Monooxygenase / Q000096:biosynthesis* / Q000235:genetics; D016133:Polymerase Chain Reaction / Q000379:methods*; D012333:RNA, Messenger / Q000096:biosynthesis*; D011994:Recombinant Proteins; D012680:Sensitivity and Specificity; D014407:Tumor Cells, Cultured
D012333:RNA, Messenger; D011994:Recombinant Proteins; D014442:Monophenol Monooxygenase; C068508:Tth polymerase; D004259:DNA-Directed DNA Polymerase