Development of tissue fixatives to minimize DNA fragmentation - DNA analysis for formalin-fixed and paraffin-embedded tissues -

为了使福尔马林作为组织固定剂对基因组DNA分子的降解最小化，EDTA，氯化钠，用10%v/v磷酸盐缓冲福尔马林溶液浸泡人体组织标本（pH7.2）和不同浓度的这些添加剂作用24小时至2年后，取5 μm厚的薄切片，用二甲苯脱蜡，提取5个薄切片的DNA。用酚-氯仿法提取DNA片段，琼脂糖凝胶电泳。将凝胶条修剪成1.2kbp大小，用紫外吸收法测定凝胶条中DNA的含量。结果表明：固定7天后，大于1.2kbp的DNA在福尔马林固定液中的扩增率为20%，在缓冲福尔马林固定液中的扩增率为42%。EDTA和黄酮类化合物可使DNA含量提高2-4%，用福尔马林单独固定后，一天后无大片段DNA残留，两年后，EDTA可使1.2kbp DNA含量提高12%，而其它添加剂均低于7%。EDTA固定液在2年后仍能为D1 S80的PCR分型提供良好的肺、肾、肝DNA模板。EDTA能显著抑制组织中DNase I的活性，其浓度为5 mM时就足够了，因此推荐使用EDTA和缓冲福尔马林作为组织固定剂。

To minimize degradation of genomic DNA molecules with formalin as the tissue fixative, the additives of EDTA, sodium chloride, methanol and flavonoid were examined.Human tissue specimen was soaked with 10%v/v phosphate-buffered formalin (pH 7.2) and various concentrations of these additives for 24 hours to two years.Thin-sectioned specimen in five μm thick was deparaffinized with xylol and the DNA of five thin-sections in every sample was extracted by means of the phenol-chloroform method.DNA samples extracted were electrophoresed in the agarose gel. Gels were trimmed at 1.2kbp in size.Contents of DNAs extracted from respective gel strips were measured by UV absorbance. Then, these DNAs were amplified by PCR for the loci of D1S80 and some STRs.The results were as follows :For 7 days after fixation, DNAs of more than 1.2kbp decreased drastically to 20% with formalin alone, and to 42% with buffered formalin. EDTA and flavonoid improved 2-4% additively.After the day, no large DNA was remained with formalin alone fixative.Two years later, EDTA addition resulted to be 12% of 1.2kbp DNA content, while other additives were less than 7%. Methanol brought to be worse.Even after 2 years, EDTA fixative gave good template of DNAs of the lung, the kidney and the liver for PCR-based genotyping of D1S80. In addition, TPOX, vWA and THO1 were possibly amplified by more than 500bp size template.EDTA inhibited significantly DNase I activity in the tissues, which concentration was enough at 5mM.Taken all together, EDTA and buffered formalin for tissue fixatives is preferentially recommended.