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/μl). 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.