A new standardized absolute quantitative RT-PCR method for detection of tyrosinase mRNAs in melanoma patients: Technical and operative instructions

Francesca Vendittelli, Concetta Santonocito, Andrea Paradisi, Federica Romitelli, Paola Concolino, Sara Lanza Silveri, Teresa Sisto, Rodolfo Capizzi, Caterina Catricalà, Antonio Mulè, Aldo Di Carlo, Cecilia Zuppi, Ettore Capoluongo

Research output: Contribution to journalArticle

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 106 to 101 copies/μl). Recovery assays for RNA and cells were also performed. Results: Our method was able to detect less than 5 cells/108 WBC and about 100 fg of tyrosinase RNA. Very low CVs (

Original languageEnglish
Pages (from-to)100-105
Number of pages6
JournalClinica Chimica Acta
Volume409
Issue number1-2
DOIs
Publication statusPublished - Nov 3 2009

Keywords

  • Melanoma markers
  • Nested PCR
  • Real-time PCR
  • Tyrosinase

ASJC Scopus subject areas

  • Biochemistry
  • Clinical Biochemistry
  • Biochemistry, medical

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    Vendittelli, F., Santonocito, C., Paradisi, A., Romitelli, F., Concolino, P., Silveri, S. L., Sisto, T., Capizzi, R., Catricalà, C., Mulè, A., Di Carlo, A., Zuppi, C., & Capoluongo, E. (2009). A new standardized absolute quantitative RT-PCR method for detection of tyrosinase mRNAs in melanoma patients: Technical and operative instructions. Clinica Chimica Acta, 409(1-2), 100-105. https://doi.org/10.1016/j.cca.2009.09.002