Sažetak
Uvod: Fiksacija tkiva formalinom, koja predstavlja široko korišćenu proceduru za konzervaciju tkiva, dovodi do oštećenja molekula DNK. Faktori koji mogu da utiču na proces fiksacije i integritet molekula DNK su brojni: dužina inkubacije, vrsta tkiva, koncentracija formalina, temperatura, pH, viskozitet.Cilj: Cilj ovog istraživanja je ispitati uticaj vremena inkubacije i temperature fiksativa na prinos, čistoću i integritet DNK izolovane iz zdravog humanog srčanog tkiva, uzetog tokom sudsko-medicinske obdukcije.
Materijal i metode: Uzorci tkiva srca su fiksirani u fosfat-puferizovanom formalinu na +4°C u mraku, kao i na sobnoj temperaturi u prisustvu svetlosti. Dezoksiribonukleinska kiselina (DNK) je izolovana nakon jednog dana, potom sukcesivno svakog dana tokom prve nedelje, zatim desetog dana, i nakon dve i četiri nedelje korišćenjem metode ekstrakcije pomoću fenol-hloroform-izoamil alkohola. Apsorbance su izmerene na 260 nm i 280 nm,
što je omogućilo izračunavanje prinosa i čistoće nukleinskih kiselina u uzorcima. Urađena je amplifikacija dva gena lančanom reakcijom polimeraze (PCR), glicerol-3-fosfat dehidrogenaze (GPDH) (150 bp) i ß-actin-a (262 bp), kako bi se procenio stepen fragmentacije molekula DNK.
Rezultati: Najveći prinos, čistoća uzoraka, kao i očuvan integritet molekula DNK dobijeni su u uzorcima tkiva koji su fiksirani u formalinu na +4°C u mraku. U ovim uzorcima GPDH i ß-actin geni su amplifikovani do 14-og dana inkubacije u formalinu, za razliku od uzoraka koji su fiksirani na sobnoj temperaturi, a u kojima je ß-actin gen amplifikovan do petog dana, a GPDH do desetog dana fiksacije.
Zaključak: Temperatura formalina i dužina fiksacije u formalinu imaju značajan uticaj na
prinos, čistoću i integritet molekula DNK.
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