The effects of tissue fixation alternatives on DNA content: a study on normal colon tissue.
The aim of this study is to investigate the effects of different fixatives on DNA, and to evaluate the fixation options for molecular studies including polymerase chain reaction (PCR) and fluorescence in-situ hybridization (FISH). Three normal-looking colonic segments from surgical resections were used for tissue sampling. The full thickness of the colonic tissues (3 mm diameter) was sampled. Tissues were fixed in 70% ethanol, 10% neutral-buffered formalin, Hollande, B5, Bouin, and Zenker solutions for 1, 2, 5, 12, 24, and 48 hours, and processed and embedded in paraffin in a standard protocol. Quantitative measurements of the extracted DNA were carried out. DNA quality was tested by PCR for the human beta globin gene. Tissue sections were also tested for the availability of FISH, by using a Her-2/neu protocol. All fixation alternatives were found to be reasonable sources of DNA for molecular studies, and they enabled the successful PCR amplification of a housekeeping gene. DNA yields were predominantly over 1000 bp in 70% ethanol and 24-hour 10% neutral-buffered formalin fixations. As for B5 and Hollande, the DNA molecules obtained were almost all smaller than 100 bp. All tissues fixed in formalin, ethanol, and Hollande were found suitable for Her-2/neu visualization after standard FISH applications, but tissues fixed in Zenker, B5, and Bouin were not found suitable.
Baloglu
Gulcan
G
Department of Biochemistry, Anatolian Health Center, Izmit, Turkey. hbaloglu@superonline.com
Haholu
Aptullah
A
Kucukodaci
Zafer
Z
Yilmaz
Ismail
I
Yildirim
Sukru
S
Baloglu
Huseyin
H
D000230:Adenocarcinoma / Q000737:chemistry / Q000235:genetics; D003106:Colon / Q000737:chemistry* / Q000378:metabolism*; D003110:Colonic Neoplasms / Q000737:chemistry / Q000235:genetics; D004247:DNA / Q000737:chemistry* / Q000378:metabolism*; D004273:DNA, Neoplasm / Q000737:chemistry / Q000378:metabolism; D005404:Fixatives; D006801:Humans; D017403:In Situ Hybridization; D016612:Paraffin Embedding; D016133:Polymerase Chain Reaction; D016707:Tissue Fixation
D004273:DNA, Neoplasm; D005404:Fixatives; D004247:DNA