Successful total RNA (totRNA) extraction is a prerequisite for a successful real-time PCR. In the present work we compared one manual and one automated totRNA extraction method for detection of Plum pox potyvirus (PPV) in leaves of different Prunus sp. using developed real-time RT-PCR assay. Advantages and disadvantages of compared methods are described in the view of sensitivity, reproducibility and in terms of laboratory use. The results suggest that the column based totRNA extraction method is more effective for apricot, plum and peach. In the case of damson the differences in real-time PCR results between both extraction methods were negligible. In case of negative results obtained with automated method, manual column based extraction method should be used additionally. Uspešnost izolacije RNA je osnova za uspešno izvedbo reakcije PCR v realnem času (qPCR). V svojem delu smo primerjali ročno in avtomatsko metodo izolacije celokupne RNA (totRNA) iz listov različnih rastlinskih vrst iz rodu Prunus. Uspešnost izolacije totRNA smo preverjali z uvedeno metodo qPCR za detekcijo virusa šarke (Plum pox potyvirus - PPV). Prednosti in slabosti uporabljenih metod smo ugotavljali s primerjavo občutljivosti detekcije, ponovljivosti in intenzivnosti laboratorijskega dela. Rezultati raziskave so pokazali, da je ročna metoda izolacije totRNA učinkovitejša pri marelicah, breskvah in slivah, medtem ko so bile razlike med obema metodama pri ciborah zanemarljive. Pri uporabi preizkušene avtomatske metode izolacije totRNA je priporočljivo, da v primeru negativnih rezultatov le-te še dodatno preverimo z uporabo ročne metode izolacije.