Malignant melanoma is a type of skin cancer that arises from melanocytes and often spreads to other organs – metastasizes, making it the major cause of death from skin cancer (more than 90% of all skin cancer-related deaths). The most rapidly developing and highly successful treatment for malignant melanoma is immunotherapy with immune checkpoint inhibitors (ICI). For predicting the effectiveness of therapy and the response to treatment with ICI, prognostic biomarkers are crucial. In this study, we established methods for determining the expression of two biomarkers in patients with malignant melanoma: IFNgama and PD-L1, from plasma and formalin-fixed paraffin-embedded (FFPE) sections of tumor tissue. We found that for evaluating gene expression in FFPE tissue, proper preparation of FFPE tissue, appropriate storage of samples, selection of suitable methods for RNA isolation from tissue, transcription into cDNA, and the selection of appropriate housekeeping genes in polymerase chain reaction (PCR) are crucial. In determining the expression of biomarkers in blood plasma, we found that cell-free RNA (cfRNA) is present in the blood at least three days after blood collection if stored at 5°C, and that reverse transcription quantitative PCR (RT-qPCR) with gene fragments gBlocks is an appropriate method for detecting cfRNA in the blood of melanoma patients. CfRNA in plasma is present in both healthy individuals and melanoma patients.
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