Evaluation of standard tyrosinase RT-PCR in melanoma patients by the use of the LightCycler system.
BACKGROUND
Haematogenous spread influences outcome in melanoma patients. The clinical relevance of detecting circulating melanoma cells in peripheral blood by tyrosinase mRNA RT-PCR is, however, questioned as rates of positivity considerably vary between studies. Standard tyrosinase-nested RT-PCR was here compared with a real-time PCR technique.


METHODS
Forty-three blood samples from 20 stage III--IV melanoma patients were analyzed. Mononuclear cells were isolated using a Ficoll Hypaque gradient technique. Total RNA extracted by the acid guanidinum thiocyanate-phenol-chloroform method was reverse transcribed using random hexamers or specific primers. Standard tyrosinase-nested PCR was performed on Touchdown machine (Hybaid) and real-time PCR on a LightCycler instrument (Roche).


RESULTS
Only two samples from stage IV patients (one from random hexamers, one from antisense primers) were found tyrosinase positive with a 100% agreement between the two PCR techniques. A 10-fold dilution of the first-round products improved the PCR kinetic and the final amount of amplified product of positive samples, but not the rate of positivity.


CONCLUSIONS
Efficiency of the PCR reaction can be monitored in an online fashion by the LightCycler instrument allowing technical improvements. However, tyrosinase mRNA RT-PCR cannot be yet considered as a useful technique in the monitoring of melanoma patients.
Department of Dermatology, National Cancer Center, Plovdivsko pole Str. No. 6, Sofia 1756, Bulgaria. kstoitchkov@hotmail.com
K
K
Stoitchkov
S
S
Letellier
J P
JP
Garnier
M
M
Toneva
E
E
Naumova
E
E
Peytcheva
N
N
Tzankov
B
B
Bousquet
P
P
Morel
T
T
Le Bricon
D000328:Adult; D000368:Aged; D005260:Female; D006801:Humans; D008297:Male; D008545:Melanoma / Q000097:blood / Q000201:enzymology*; D008875:Middle Aged; D012333:RNA, Messenger / Q000097:blood / Q000235:genetics; D020133:Reverse Transcriptase Polymerase Chain Reaction; D012878:Skin Neoplasms / Q000097:blood / Q000201:enzymology*; D014443:Tyrosine / Q000097:blood* / Q000235:genetics
D012333:RNA, Messenger; D014443:Tyrosine