title: Quantitative assessment of the expression of melanoma-associated antigens by non-competitive reverse transcription polymerase chain reaction.
abstract: The assessment of tumor-associated antigens (TAA) recognized by T lymphocytes is a prerequisite for diagnosis and immunotherapy of melanoma. Different reverse transcription-polymerase chain reaction (RT-PCR) protocols allowing the quantification of the TAA mRNA expression in the solid tumor or the detection of circulating melanoma cells have been described. We have recently shown a positive correlation between the amount of specific product formed 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 ethidium bromide-stained agarose gels, followed by computer-assisted densitometry. To standardize our method, we examined the linear range of the densitometric quantification procedure as reflected by the correlation of signal intensity to the amount of the corresponding DNA. As an internal measure for the so-termed cDNA in the different samples after RNA isolation and reverse transcription, a beta-actin PCR was introduced. Subsequently, we chose four sets of primers for the melanoma-associated antigens MAGE1, tyrosinase, Melan A/MART-1 and gp100/Pmel17 and performed PCR analysis over a range of cycle numbers. In each case, the amplification rate remained constant 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 summary, the method introduced in the present work allows the quantification of TAA in melanoma which might be important for the monitoring of disease. Technically the method is sound and sensitive, avoids post-PCR manipulations and can be performed with the standard equipment of a molecular biology laboratory. It can be applied also to other solid tumors and leukemias.
authors.affiliation: Third Department of Medicine, University of Ulm, D-89081 Ulm, Germany.
authors.forename: M
authors.initials: M
authors.lastname: Ringhoffer
authors.forename: M
authors.initials: M
authors.lastname: Schmitt
authors.forename: J
authors.initials: J
authors.lastname: Karbach
authors.forename: E
authors.initials: E
authors.lastname: Jäger
authors.forename: F
authors.initials: F
authors.lastname: Oesch
authors.forename: M
authors.initials: M
authors.lastname: Arand
mesh_terms: D000951:Antigens, Neoplasm / Q000235:genetics* / Q000378:metabolism; D017931:DNA Primers / Q000737:chemistry; D006801:Humans; D058965:MART-1 Antigen; D008545:Melanoma / Q000235:genetics / Q000378:metabolism*; D058950:Melanoma-Specific Antigens; D008562:Membrane Glycoproteins / Q000235:genetics / Q000378:metabolism; D014442:Monophenol Monooxygenase / Q000235:genetics / Q000378:metabolism; D009363:Neoplasm Proteins / Q000235:genetics* / Q000378:metabolism; D012333:RNA, Messenger / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D012680:Sensitivity and Specificity; D012878:Skin Neoplasms / Q000235:genetics / Q000378:metabolism*; D058951:gp100 Melanoma Antigen
chemical_list: D000951:Antigens, Neoplasm; D017931:DNA Primers; C072011:MAGEA1 protein, human; D058965:MART-1 Antigen; C548667:MLANA protein, human; D058950:Melanoma-Specific Antigens; D008562:Membrane Glycoproteins; D009363:Neoplasm Proteins; C548637:PMEL protein, human; D012333:RNA, Messenger; D058951:gp100 Melanoma Antigen; D014442:Monophenol Monooxygenase