FORMALIN FIXATION AT LOW TEMPERATURE PROVIDES BETTER YIELD AND INTEGRITY OF DNA
Abstract
Introduction: Fixation with formalin, a widely used procedure to preserve tissue samples, leads to damage of DNA through crosslinking activity. The factors that could influence the fixation and integrity of DNA may be numerous: incubation time, tissue type, concentration
of formalin, temperature, pH and viscosity.
Aim: The aim of this investigation was to examine the influence of incubation time and temperature of formalin fixation on the yield, purity and integrity of DNA isolated from healthy
human heart myocardial tissue taken during medico-legal autopsy.
Material and methods: Heart tissue samples were fixed in phosphate-buffered formalin at +4°C in the dark, as well as at room temperature in the presence of light. The DNA was isolated after one day, then successively every day during the first week, and then on the tenth day, and after two and four-week periods using phenol-chloroform-isoamyl alcohol extraction method. The absorbances were measured at 260 nm and 280 nm, which allows calculation of yield and purity of nucleic acid in the samples. The PCR amplification of two genes, GPDH (150 bp) and ß-actin (262 bp), were performed to evaluate the degree of DNA molecule fragmentation.
Results: The highest yield, purity and preserved integrity of DNA were obtained from the samples fixed in formalin at +4°C in the darkness. In these samples, GPDH and ß-actin genes
were amplified up to 14 days, unlike the samples that were fixed at the room temperature in which the ß-actin gene was amplified up to 5 days, while the GPDH gene fragment was successfully amplified up to 10 days of fixation.
Conclusion: The temperature, presence of light and the incubation time of formalin fixation all have important influences on yield, purity and integrity of DNA during the fixation process.
References
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.