MART-1 mRNA and the RNA internal standard were amplified by reverse transcription followed by two rounds of PCR amplification using two sets of MART-1-specific primers as outlined in Fig.
SK-MEL-28 cells (10 000) diluted in 1 mL of MART-1 mRNA-negative blood were used to prepare the calibrators.
The unknown samples and a dilution series of the calibrator RNA covering the range 1.5-150 x [10.sup.-21] mol of MART-1 mRNA/mL were incubated with RNA internal standard, and the nested MART-1 RT-PCR reaction was performed.
QUANTIFICATION OF MART-1 mRNA IN BLOOD FROM PATIENTS WITH MALIGNANT MELANOMA AND CONTROLS
The purified RNA was subjected to the quantitative MART-1 mRNA assay.