20.500.12556/RUL-154487 Validacija metode za izolacijo RNA iz tumorjev in prostocelične RNA iz plazme rakavih bolnikov Validation of a method for isolation of RNA from tumours and cell-free RNA from plasma of cancer patients Melanom je oblika kožnega raka, ki nastane v melanocitih in pogosto zaseva, zato je vzrok za več kot 90 % vseh smrti zaradi kožnega raka. Najhitreje razvijajoče se in zelo uspešno zdravljenje malignega melanoma je imunoterapija z zaviralci imunskih kontrolnih točk (ZIKT). Za napovedovanje učinkovitosti terapije in odgovora na zdravljenje z ZIKT pa so zelo pomembni biomarkerji pri posameznem bolniku. V nalogi smo vzpostavili metodi za določanje izražanja dveh biomarkerjev pri bolnikih z malignim melanomom, in sicer IFNgama in PD-L1 iz plazme in iz rezin tumorskega tkiva, fiksiranega s formalinom in vklopljenega v parafin (FFPE tkivo). Ugotovili smo, da je za ugotavljanje izražanja genov v FFPE tkivu zelo pomembna ustrezna priprava FFPE tkiva, primerno shranjevanje vzorcev, izbira ustreznih metod za izolacijo RNA iz tkiva in prepis v cDNA ter izbor ustreznih hišnih genov pri verižni reakciji s polimerazo (PCR). Pri določanju izražanja biomarkerjev v krvni plazmi smo ugotovili, da je prostocelična RNA (cfRNA) v krvi prisotna vsaj tri dni po odvzemu krvi, če je le-ta skladiščena pri 5 °C, in da je verižna reakcija s polimerazo z revezno transkripcijo (RT-qPCR) z genskimi fragmenti gBlocks ustrezna metoda za dokazovanje cfRNA v krvi bolnikov z melanomom. CfRNA v plazmi je prisotna tako pri zdravem posamezniku kot pri bolniku z melanomom. 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. melanom imunoterapija ZIKT biomarkerji IFNgama PD-L1 RNA FFPE tkivo krvna plazma melanoma immunotherapy ICI biomarkers IFNgama PD-L1 RNA FFPE tissue blood plasma true false false [M. Kerin] Slovenski jezik Angleški jezik Magistrsko delo/naloga 2024-02-17 07:15:19 2024-02-17 07:15:26 2024-02-26 10:42:09 0000-00-00 00:00:00 2024 0 0 0000-00-00 NiDoloceno NiDoloceno NiDoloceno 0000-00-00 0000-00-00 0000-00-00 1970-01-01 616-006.81:575(043.2) 236268 186738947 38418.pdf 38418.pdf 1 5C582EEBEFE992F5965D7CDEB1449065 db33a1a5614e21d426b25a7f98651982055e6bb7188f4dc229d11de90c8f06ea fd3ff18b-cd5b-11ee-a3c7-0050569b8976 https://repozitorij.uni-lj.si/Dokument.php?lang=slv&id=180947 Biotehniška fakulteta 0 0 0