Tian Zi-Qiang, Liu Jun-Feng, Zhang Shao-Wei, Li Bao-Qing, Wang Fu-Shun, Zhang Yue-Feng
Department of Thoracic Surgery, Fourth Hospital of Hebei Medical University, Shijiazhuang, Hebei, 050011, PR China.
Ai Zheng. 2004 Mar;23(3):342-435.
BACKGROUND & OBJECTIVE: Unbuffered formalin is widely used to fix resected specimens in China. The DNA in unbuffered formalin-fixed and paraffin-embedded tissues is usually degraded seriously, so the extraction of DNA from these samples is difficult. This study was conducted to seek an optimal method to extract DNA from these samples.
Fifteen blocks of esophageal carcinoma resected in Fourth Hospital of Hebei Medical University in 2000 were selected. The cells were lyzed by proteinase K digestion or heating under different pH values, then DNA was extracted by phenol:chloroform. After that, four parameters (deparaffined by xylene or histolene; digested for 48 h or 72 h at 37 degrees C or 56 degrees C; extracted by salting-out or phenol:chloroform) were optimized according to the principle of cross design. At last, the quality of obtained DNA was analyzed with electrophoresis and PCR amplification.
The quality and quantity of DNA obtained by proteinase K digestion (the average yield is 17.88 microg) were better than that of heating under different pH (7-12)(P< 0.05). The quality and quantity of DNA digested at 56 degrees C were better than that at 37 degrees C, and similarly, digestion for 72 hours was better than that for 48 hours. The methods of deparaffin and extraction had no obvious influence on the quality and quantity of DNA.
By means of NaCl salting-out after proteinase K digestion, more reliable quality of DNA can be obtained from unbuffered formalin-fixed and paraffin-embedded samples. Furthermore,digestion for three days at 56 degrees C is more likely to obtain DNA with high quality and quantity.
在中国，未缓冲的福尔马林被广泛用于固定切除的标本。未缓冲的福尔马林固定石蜡包埋组织中的DNA通常严重降解，因此从这些样本中提取DNA很困难。本研究旨在寻找从这些样本中提取DNA的最佳方法。
选取2000年在河北医科大学第四医院切除的15例食管癌组织块。通过蛋白酶K消化或在不同pH值下加热裂解细胞，然后用苯酚：氯仿提取DNA。之后，根据交叉设计原则对四个参数（用二甲苯或组织透明剂脱石蜡；在37℃或56℃消化48小时或72小时；用盐析法或苯酚：氯仿提取）进行优化。最后，用电泳和PCR扩增分析获得的DNA质量。
通过蛋白酶K消化获得的DNA质量和数量（平均产量为17.88微克）优于在不同pH值（7-12）下加热获得的DNA（P<0.05）。在56℃消化的DNA质量和数量优于在37℃消化的，同样，消化72小时优于消化48小时。脱石蜡和提取方法对DNA的质量和数量没有明显影响。
通过蛋白酶K消化后用氯化钠盐析法，可以从未缓冲的福尔马林固定石蜡包埋样本中获得质量更可靠的DNA。此外，在56℃消化三天更有可能获得高质量和数量的DNA。
