title: A new standardized absolute quantitative RT-PCR method for detection of tyrosinase mRNAs in melanoma patients: technical and operative instructions.
abstract: BACKGROUND
  To develop a new absolute quantitative real-time PCR method for blood mRNA tyrosinase assay and to compare this new method with standard RT-PCR nested.
  
  
  METHODS
  Ten blood of melanoma patients (stages I-III), 5 tissue samples, 2 surgical fresh metastatic skin and 3 lymph nodes paraffin-embedded slices were analysed, and 10 negative controls were used. Ten millilitres of blood was analysed for each individual. Three different protocols for RNA extraction and two reverse transcription methods were used. Specific human tyrosinase cDNA fragment was cloned into pcDNA3+ vector and then titrated for the standard curve construction (from 10(6) to 10(1)copies/microl). Recovery assays for RNA and cells were also performed.
  
  
  RESULTS
  Our method was able to detect less than 5 cells/10(8) WBC and about 100 fg of tyrosinase RNA. Very low CVs (<1.5%) were obtained on all samples run in triplicate. Sensitivity and specificity were of 100%. The amount of starting volume of blood was crucial for the determination of copy number since large volumes are necessary for patient's monitoring.
  
  
  CONCLUSIONS
  Our absolute qRT-PCR assay could be proposed as a new standardized molecular method for the management of melanoma patients, particularly for the follow up of the highest AJCC stages.
authors.affiliation: Laboratory of Clinical Molecular Biology, Department of Biochemistry & Clinical Biochemistry, Catholic University, Rome, Italy.
authors.forename: Francesca
authors.initials: F
authors.lastname: Vendittelli
authors.forename: Concetta
authors.initials: C
authors.lastname: Santonocito
authors.forename: Andrea
authors.initials: A
authors.lastname: Paradisi
authors.forename: Federica
authors.initials: F
authors.lastname: Romitelli
authors.forename: Paola
authors.initials: P
authors.lastname: Concolino
authors.forename: Sara Lanza
authors.initials: SL
authors.lastname: Silveri
authors.forename: Teresa
authors.initials: T
authors.lastname: Sisto
authors.forename: Rodolfo
authors.initials: R
authors.lastname: Capizzi
authors.forename: Caterina
authors.initials: C
authors.lastname: Catricalà
authors.forename: Antonio
authors.initials: A
authors.lastname: Mulè
authors.forename: Aldo
authors.initials: A
authors.lastname: Di Carlo
authors.forename: Cecilia
authors.initials: C
authors.lastname: Zuppi
authors.forename: Ettore
authors.initials: E
authors.lastname: Capoluongo
mesh_terms: D016022:Case-Control Studies; D005260:Female; D006801:Humans; D007753:Laboratories; D008207:Lymphatic Metastasis; D008297:Male; D008545:Melanoma / Q000097:blood / Q000235:genetics* / Q000473:pathology; D014442:Monophenol Monooxygenase / Q000235:genetics*; D009367:Neoplasm Staging; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D015203:Reproducibility of Results; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods* / Q000592:standards*; D048348:Reverse Transcription; D012878:Skin Neoplasms / Q000097:blood / Q000556:secondary
chemical_list: D012333:RNA, Messenger; D014442:Monophenol Monooxygenase