Določanje tirozinazne mRNK s postopkom RT-PCR v periferni krvi bolnikov z malignim melanomom
Glumac, Nebojša
OUTLINE: Malignant melanoma (MM) is one of the most aggressive cancers in humans. It has a high metastatic potential even in small primary lesions. Unfortunately, the incidence of this skin cancer is ...increasing by 10% per year. The differentiation between localised and advanced disease is currently based on cytological verification of malignant cells in clinically suspicious regional lymph nodes. The use of this method is limited in the early detectionof micrometastases, and absolutely unusable in the detection of circulating melanoma cells having a metastatic potential. Therefore, the objective of our study is to develop a sensitive RT-PCR assay for the detection of tyrosinase mRNA and, hence, to detect circulating melanoma cells in full venous blood of patients with MM. We are exploiting the fact that tyrosinase is a tissue specific enzyme which is only expressed in normal skin melanocytes and MM cells that invade blood during tnetastasing. METHODS AND MATERIALS: Eighteen patients with American Joint Committee on Cancer stage IIIand IV disease and 8 healthy volunteers were included in our study. Ten ml of peripheral blood was taken from each subject and total RNA was isolated using acid guanidium isothiocyanate, phenol and chloroform. Total RNA was transcribed into cDNA using enzyme reverse transcriptase (RT) and random hexameres as primers. The product of previous reaction was multiplied in nested polymerase chain reaction (PCR) using tyrosinase specific set of primers. The expression of ubiquitously present gene for glyceraldehyde 3-phosphate dehydrogenase was used as a control for the reaction. The final products were analysed on 2% agarose gels and stained with ethidium bromide. RESULTS: After intensive optimising of the whole procedure, the lowest detection limit of 2500 B-16 MM cells cultured in vitro was achieved. (Abstract truncated at 2000 characters).
Type of material - research report
Publication and manufacture - Ljubljana : [N.Glumac], 2000
Prešernove naloge GLUMAC Nebojša Določanje IN: 020010381
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