Quantitative assessment of the expression of melanoma-associated antigens by non-competitive reverse transcription polymerase chain reaction

The assessment of tumor-associated antigens (TAA) recognized by T lymphocyt es is a prerequisite for diagnosis and immunotherapy of melanoma. Different reverse transcription-polymerase chain reaction (RT-PCR) protocols allowin g the quantification of the TAA mRNA expression in the solid tumor or the d etection of circulating, melanoma cells have been described. We have recent ly shown a positive correlation between the amount of specific product form ed by RT-PCR and the staining intensity in immunohistochemical analysis of the corresponding sample. Here we describe a quantification procedure based on the direct digitization of the PCR products after separation on ethidiu m bromide-stained agarose gels, followed by computer-assisted densitometry. To standardize our method, we examined the linear range of the densitometr ic quantification procedure as reflected by the correlation of signal inten sity to the amount of the corresponding DNA. As an internal measure for the so-termed 'effective' CDNA in the different samples after RNA isolation an d reverse transcription, a beta -actin PCR was introduced. Subsequently, we chose four sets of primers for the melanoma-associated antigens MAGE1, tyr osinase, Melan A/MART-1 and gp100/Pmel17 and performed PCR analysis over a range of cycle numbers. In each case, the amplification rate remained const ant up to at least 26 cycles under the respective conditions. Plotting the logarithm of the amount of product against the cycle number yields a slope that equals the logarithm of the amplification rate. The amount of starting material can be determined from the intercept with the ordinate. In summar y, the method introduced in the present work allows the quantification of T AA in melanoma which might be important for the monitoring of disease. Tech nically the method is sound and sensitive, avoids post-PCR manipulations an d can be performed with the standard equipment of a molecular biology labor atory. It can be applied also to other solid tumors and leukemias.