比较70％乙醇与中性甲醛固定对肺癌组织DNA质量影响的实验研究

目的由于常规10％中性甲醛固定的组织DNA提取质量较低，易发生断裂或降解，不利于保存及长片段DNA扩增。本研究比较70％乙醇与中性甲醛固定法对DNA质量及后续PCR的影响。方法取70％乙醇固定的肺癌组织30例（其中10例固定7天以上），10％中性甲醛固定的自身配对癌组织30份。采用试剂盒提取DNA，分析条带完整性和纯度，并用PCR扩增HBB（beta globin）及SERPINA9（antitrypsin）两基因的不同长度片段，用琼脂糖凝胶电泳分析扩增产物。结果70％乙醇固定组的基因组DNA经电泳条带整齐未见明显降解，而10％中性甲醛组DNA条带弥散，有可见降解。后续普通PCR扩增268bp、536bp的产物显示乙醇固定组产量明显高于10％中性甲醛固定组。实时荧光定量PCR也显示在扩增123bp、251bp、346bp三个不同长度片段时乙醇固定组产量均明显高于10％中性甲醛固定组（P〈0．01）。中性甲醛组DNA在扩增346bp长度片段时成功率下降至47％（14／30）。乙醇固定时间长短对DNA的antitrypsin模板质量无明显差异。结论70％乙醇固定法对肺癌组织DNA保存效果优于中性甲醛法，且更适合长片段DNA扩增。长时间乙醇同定法对DNA质量无明显影响，可适合于长途运输生物标本。

Experimental study on the quality comparison of DNA samples from lung cancer tissues fixed by 70% ethanol versus neutral formalin

Objective Owing to possible DNA breakage and degradation, tissues fixed by 10% neutral formalin may not be suitable for long - term preservation and downstream long DNA fragment amplification. This study aimed to compare the DNA quality extracted from lung cancer samples fixed by 70% ethanol versus neutral formalin. Methods Thirty cases of lung cancer tissues by 70% ethanol fixation（ 10 of which were collected by long time fixation more than 7 days）and 30 cases of formalin fixed samples were subject to DNA extraction with ＂Tissue/Cell DNA Extract kit＂. Then DNA yields and purity were analyzed by agarose gel electrophoresis. Different length of expected DNA fragments of genes HBB（ beta hemaglobin）and SERPINA9（antitrypsin） were amplilied by common PCR and real - time quantitative PCR. Yields of PCR products were also compared by agarose gel electrophoresis. Results The integrity of DNAs extracted in 70% ethanol tissues was good without observed degradation, while DNAs from 10% formalin samples showed obvious smear bands during electrophoresis. Following common PCR, both yields of expected bands of 268 bp and 536 bp were more seen in group of 70% ethanol than in formalin group. By real time qPCR,all three amplicons of 100 bp,250 bp and 350 bp were more produced and shown at lower Ct values in 70% ethanol group compared with formalin group（ P 〈0.01 ）. The success rate of qPCR for 346bp amplicons in formalin group samples was only 47% （14/30）. DNA templates of antitrypsin were not significantly influenced by duration of ethanol fixation. Conclusion Quality of DNAs from 70% ethanol - fixed specimens was superior to formalin - fxed samples and suitable for long fragment amplification. Ethanol -fixed samples may be also suitable for long time transportatien without compromising the DNA quality.