RT-PCR detection of tyrosinase, gp100, MART1/Melan-A, and TRP-2 gene transcripts in peripheral blood of melanoma patients.
OBJECTIVE
To detect the expression of genes encoding tyrosinase, gp100, MART-1/Melan A, and tyrosinase-related protein-2 (TRP-2) in peripheral blood of melanoma patients by reverse transcription-polymerase chain reaction (RT-PCR).


METHODS
Nineteen peripheral blood samples were obtained from 17 melanoma patients. When tested, 15 of them presented with clinically detectable metastatic disease. Samples of peripheral blood (7 mL) were collected from each patient into vacutainer cell preparation tubes. Mononuclear cells were isolated, total cellular RNA extracted, and then used as a template for reverse transcription to complementary DNA (cDNA). The cDNA was thereafter assayed by PCR for the expression of melanocyte-associated transcripts of tyrosinase, gp100, MART1/Melan-A, and TRP-2 genes.


RESULTS
Gp100 gene expression was detected in 13 out of 19 samples. In 4 of them, TRP-2 gene expression was also detectable. Expression of tyrosinase and Melan-A/MART-1 genes could not be observed. Interestingly, gp100 and TRP-2 gene transcripts were detected in patients having recurrent and/or metastatic disease at the time of testing.


CONCLUSIONS
The results we obtained support the use of RT-PCR assay for indirect detection of melanoma cells in peripheral blood of melanoma patients. As the transcripts for the tyrosinase gene and MART-1/Melan A gene were not detected, additional optimization experiments of RT-PCR assay are required.
Samija
M
M
University Hospital for Tumors, Zagreb, Croatia.
Juretić
A
A
Solarić
M
M
Samija
I
I
Bingulac-Popović
J
J
Grahovac
B
B
Stanec
M
M
Oresić
V
V
D000328:Adult; D000368:Aged; D000939:Epitopes / Q000097:blood / Q000235:genetics; D005260:Female; D015870:Gene Expression; D006801:Humans; D019746:Intramolecular Oxidoreductases / Q000097:blood / Q000235:genetics; D008297:Male; D008545:Melanoma / Q000235:genetics*; D008562:Membrane Glycoproteins / Q000097:blood / Q000235:genetics; D008875:Middle Aged; D009363:Neoplasm Proteins / Q000097:blood / Q000235:genetics; D020133:Reverse Transcriptase Polymerase Chain Reaction; D012680:Sensitivity and Specificity; D012878:Skin Neoplasms / Q000235:genetics*; D058951:gp100 Melanoma Antigen
D000939:Epitopes; C105787:MART-1-Melan-A(27-35) epitope; D008562:Membrane Glycoproteins; D009363:Neoplasm Proteins; C548637:PMEL protein, human; D058951:gp100 Melanoma Antigen; D019746:Intramolecular Oxidoreductases; C042501:dopachrome isomerase