Baloglu Gulcan, Haholu Aptullah, Kucukodaci Zafer, Yilmaz Ismail, Yildirim Sukru, Baloglu Huseyin
Department of Biochemistry, Anatolian Health Center, Izmit, Turkey.
Appl Immunohistochem Mol Morphol. 2008 Oct;16(5):485-92. doi: 10.1097/PAI.0b013e31815dffa6.
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.
本研究的目的是调查不同固定剂对DNA的影响，并评估用于包括聚合酶链反应（PCR）和荧光原位杂交（FISH）在内的分子研究的固定方法。取自手术切除的三个外观正常的结肠段用于组织采样。采集结肠组织的全层（直径3毫米）。将组织分别置于70%乙醇、10%中性缓冲福尔马林、霍兰德固定液、B5固定液、布因固定液和岑克尔固定液中固定1、2、5、12、24和48小时，然后按照标准方案进行处理并包埋于石蜡中。对提取的DNA进行定量测量。通过PCR检测人β珠蛋白基因来测试DNA质量。还通过使用Her-2/neu方案对组织切片进行FISH可用性检测。发现所有固定方法都是用于分子研究的合理DNA来源，并且它们能够成功地对管家基因进行PCR扩增。在70%乙醇和24小时10%中性缓冲福尔马林固定中，DNA产量主要超过1000 bp。至于B5和霍兰德固定液，获得的DNA分子几乎都小于100 bp。发现在标准FISH应用后，所有用福尔马林、乙醇和霍兰德固定的组织都适合进行Her-2/neu可视化，但用岑克尔、B5和布因固定的组织不适合。
