改良醋酸铵盐析法提取福尔马林固定石蜡包埋组织DNA

The improved ammonium acetate method in the application of DNA extraction from formalin-fixed and paraffin-embedded(FFPE) tissues

目的采用改良醋酸铵盐析法提取福尔马林固定石蜡包埋组织DNA,并评价其应用价值。方法取死后24h内人体肾组织用10%中性福尔马林溶液固定7d,石蜡包埋。采用酚/氯仿法、Chelex-100法及改良醋酸铵盐析法提取DNA。经用紫外分光光度计法、AGE及PCR-PAGE检测比较不同方法提取DNA的质量。结果改良醋酸铵盐析法、酚/氯仿法、Chelex-100法OD260/OD280比值分别为1.962±0.195、2.110±0.470、1.018±0.124,两两比较均有显著性差异(P<0.05);3种方法提取DNA含量(μg)分别为0.515±0.447、0.328±0.345、5.346±1.994;AGE电泳图谱可印证上述结果;PCR-PAGE检测显示改良醋酸铵盐析法提取DNA的谱带清晰度好于其它两种方法。结论改良醋酸铵盐析法更适合微量福尔马林固定石蜡包埋组织样本DNA的提取。

Objective The modified ammonium acetate method was used to extract DNA from the formalin-fixed and paraffin-embedded （FFPE） tissues and its application was evaluated. Methods The human kidney tissues were obtained by autopsy within 24 hours post-death and then were embedded in paraffin after fixing with 10% neutral formalin solution for 7 days. The DNA from FFPE tissues were extracted by the methods of modified ammonium acetate, phenol/chloroform, and Chelex-100. The quality and the quantity of the DNA extracted by three different methods were evaluated by UV spectrophotometer, AGE and PCR- PAGE. Results The ratio of OD26o/0D2so in modified ammonium acetate method was 1. 962± 0. 195. There was a significant difference （p 〈 0. 05 ）, in comparison with the ratio in phenol/chloroform （2. 110 ± 0. 470） and Chelex-100 （ 1. 018 ±0. 124）. The yields （μg） of DNA extracted by three different methods were 0. 515 ± 0. 447, 0. 328 ± 0. 345 and 5. 346±~ 1. 994, separately. Furthermore, the above results were confirmed by AGE and the PCR-PAGE. DNA extracted by the modified ammonium acetate method showed more clear bands than that by other methods in both of AGE and PCR-PAGE. Conclusion The modified ammonium acetate method is more suitable for DNA extracted from the micro-samples of formalin-fixed and paraffin-embedded tissues.