FIKSACIJA FORMALINOM NA NISKOJ TEMPERATURI OBEZBEĐUJE BOLJI PRINOS I INTEGRITET DNK
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.
Reference
Wienroth M, Morling N, Williams R. Technological innovations in forensic genetics: social, legal and ethical aspects. Recent Adv DNA Gene Seq. 2014; 8(2):98-103
Hamazaki S, Koshiba M, Habuchi T, Takahashi R, Sugiyama T. The effect of formalin fixation on restriction endonuclease digestion of DNA and PCR amplification. Pathol Res Pract. 1993; 189(5):553-557.
Koshiba M, Ogawa K, Hamazaki S, Sugiyama T, Ogawa O, Kitajima T. The Effect of Formalin Fixation on DNA and the Extraction of High-molecular-weight DNA from Fixed and Embedded Tissues. Path. Res. Pract. 1993; 189:66-72.
Okello JB, Zurek J, Devault AM, Kuch M, Okwi AL, Sewankambo NK, et al., editors. Comparison of methods in the recovery of nucleic acids from archival formalin-fixed paraffin-embedded autopsy tissues. Anal Biochem. 2010; 400(1):110-117.
Hewitt SM, Lewis FA, Cao Y, Conrad RC, Cronin M, Danenberg KD, et al., editors. Tissue handling and specimen preparation in surgical pathology:issues concerning the recovery of nucleic acids from formalin-fixed, paraffin-embedded tissue. Arch Pathol Lab Med. 2008; 132(12):1929-1935.
Bonin S, Petrera F, Niccolini B, Stanta G. PCR analysis in archival postmortem tissues. J Clin Pathol: Mol Pathol. 2003; 56:184-186.
Legnard B, Mazancourt P, Durigon M, Khalifat V, Crainic K. DNA genotyping of unbuffered formalin fixed paraffin embedded tissues. Forensic Sci Int. 2002; 125:205-11.
Foss RD, Guha-Thakurta N, Conran RM, Gutman P. Effects of fixative and fixation time on the extraction and polymerase chain reaction amplification of RNA from paraffin-embedded tissue. Comparison of two housekeeping gene mRNA controls. Diagn Mol Pathol. 1994; 3:148–155.
Groelz D, Sobin L, Branton P, Compton C, Wyrich R, Rainen L. Non-formalin fixative versus formalin-fixed tissue: a comparison of histology and RNA quality. Exp Mol Pathol. 2013; 94(1):188-194.
Fox CH, Johnson FB, Whiting J, Roller PP. Formaldehyde fixation. J Histochem Cytochem. 1985; 33:845-853.
Duval K, Aubin RA, Elliott J, Gorn-Hondermann I, Birnboim HC, Jonker D, et al., editors. Optimized manual and automated recovery of amplifiable DNA from tissues preserved in buffered formalin and alcohol-based fixative. Forensic Sci Int Genet. 2010; 4(2):80-88.
Bussolati G, Annaratone L, Medico E, D’Armento G, Sapino A. Formalin fixation at low temperature better preserves nucleic acid integrity. PLoS One. 2011; 6(6):e21043.
Bonin S, Stanta G. Nucleic acid extraction methods from fixed and paraffin-embedded tissues in cancer diagnostics. Expert Rev Mol Diagn. 2013; 13(3):271-82.
Srinivasan M, Sedmak D, Jewell S. Effect of fixatives and tissue processing on the content and integrity of nucleic acids. Am J Pathol. 2002 Dec; 161(6):1961-71.
Funabashi KS, Barcelos D, Visoná I, e Silva MS, e Sousa MLAPO, de Franco MF, et al. DNA extraction and molecular analysis of non-tumoral liver, spleen, and brain from autopsy samples: the effect of formalin fixation and paraffin embedding, Pathol Res Pract. 2012; 208:584–591.
Niland EE, McGuire A, Cox MH, Sandusky GE. High quality DNA obtained with an automated DNA extraction method with 70+ year old formalin-fixed celloidin-embedded (FFCE) blocks from the indiana medical history museum. Am J Transl Res. 2012; 4(2):198-205.
Start RD, Layton CM, Cross SS, Smith JH. Reassessment of the rate of fixative diffusion. J Clin Pathol 1992; 45:1120–1121.
Werner M, Chott A, Fabiano A, Battifora H. Effect of formalin tissue fixation and processing on immunohistochemistry, Am J Surg Pathol. 2000; 24:1016– 1019.