title: Comparison between three molecular methods for detection of blood melanoma tyrosinase mRNA. Correlation with melanoma stages and S100B, LDH, NSE biochemical markers.
abstract: BACKGROUND
  The molecular monitoring of circulating tumor cells by reverse transcriptase-PCR (RT-PCR) for patients with melanoma, is still under debate. It may be affected by: a) pre-analytical variability, b) frequency of melanoma-associated gene transcripts and c) the reliability of the methods employed. Few commercial methods are available for the detection of tyrosinase mRNA in blood.
  
  
  OBJECTIVE
  Comparison between two RT-PCR-nested methods with a third one based on real-time methodology, for detection and quantitation of tyrosinase transcripts, respectively.
  
  
  METHODS
  Sixty-two melanoma patients with different AJCC stages and 20 healthy subjects were enrolled. All blood samples were extracted in duplicate with two different methods. Two nested-PCR methods (one commercial and one in house) plus a real time commercial kit were employed.
  
  
  RESULTS
  The two nested PCR methods employed were overimposable, specific and sensitive, at least in the stage III, where there was a concordance between sentinel lymph nodes detection and blood tyrosinase positivity. The different extraction methods did not affect the quality of results, while the commercial real-time kit cannot be used.
  
  
  CONCLUSIONS
  Tyrosinase mRNA detection may be therefore employed to monitor the melanoma patients over time in function of response to therapy.
authors.affiliation: Laboratory of Molecular Biology, Department of Biochemistry and Clinical Biochemistry - Catholic University, Rome, Italy.
authors.forename: Concetta
authors.initials: C
authors.lastname: Santonocito
authors.forename: Paola
authors.initials: P
authors.lastname: Concolino
authors.forename: Maria Michela
authors.initials: MM
authors.lastname: Lavieri
authors.forename: Franco
authors.initials: F
authors.lastname: Ameglio
authors.forename: Stefano
authors.initials: S
authors.lastname: Gentileschi
authors.forename: Rodolfo
authors.initials: R
authors.lastname: Capizzi
authors.forename: Sandro
authors.initials: S
authors.lastname: Rocchetti
authors.forename: Pierluigi
authors.initials: P
authors.lastname: Amerio
authors.forename: Massimo
authors.initials: M
authors.lastname: Castagnola
authors.forename: Cecilia
authors.initials: C
authors.lastname: Zuppi
authors.forename: Ettore
authors.initials: E
authors.lastname: Capoluongo
mesh_terms: D014408:Biomarkers, Tumor / Q000235:genetics / Q000378:metabolism; D005260:Female; D006801:Humans; D007770:L-Lactate Dehydrogenase / Q000378:metabolism*; D008297:Male; D008545:Melanoma / Q000097:blood / Q000235:genetics / Q000378:metabolism* / Q000473:pathology*; D014442:Monophenol Monooxygenase / Q000235:genetics*; D009367:Neoplasm Staging; D009414:Nerve Growth Factors / Q000235:genetics / Q000378:metabolism*; D010751:Phosphopyruvate Hydratase / Q000235:genetics / Q000378:metabolism*; D012333:RNA, Messenger / Q000097:blood* / Q000235:genetics; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods; D064568:S100 Calcium Binding Protein beta Subunit; D009418:S100 Proteins / Q000235:genetics / Q000378:metabolism*; D012680:Sensitivity and Specificity
chemical_list: D014408:Biomarkers, Tumor; D009414:Nerve Growth Factors; D012333:RNA, Messenger; D064568:S100 Calcium Binding Protein beta Subunit; D009418:S100 Proteins; C579160:S100B protein, human; D007770:L-Lactate Dehydrogenase; D014442:Monophenol Monooxygenase; D010751:Phosphopyruvate Hydratase