染色质免疫共沉淀技术在前列腺癌细胞株中的优化

Optimation of chromatin immunoprecipitation technology in prostate cancer cells

目的优化染色质免疫共沉淀技术（ChIP）的关键条件，以期提高实验效率。方法拟以前列腺癌细胞株PC-3、DUl45为实验材料，主要对裂解液体积、超声波的功率、脉冲及工作时间等方面进行调整，超声波破碎仪设置不同的条件（25％或30％功率，35s或50s间隔，3s或5s工作时间），然后解交联富集这些DNA片段，测定其浓度及纯度值，分别行聚合酶链反应（PCR）并用2％琼脂糖凝胶电泳检测结果。结果前列腺癌细胞株合适的ChIP实验条件为：（1）超声波最大功率为30％，设定每38s行3s或55s行5s超声工作时间，总的时间45～75s；（2）纯化后的DNA浓度〉10ng／μl、纯度值（A260/A280）〉1．45，可以得到比较好的实验结果。结论在前列腺癌细胞株PC-3、DU145中，选定合适的超声波频率后，总的超声工作时间为60s时，便可得到合适的DNA片段；洗脱液体积固定后，分2次离心要比1次离心所得到的DNA纯度值高，所得到的实验结果也越明显。

Objective To optimize the key conditions of chromatin immunoprecipitation （CHIP） technology so as to improve the efficiency of the experiments. Methods Two prostate cancer cell lines （ PC - 3, and DU145） were chosen as the experimental materials, and some respects of volume of cracking liquid, ultrasonic power, pulse and working time were adjusted. Following conditions were set up： 25% or 30% maximum power, 35 s or 50 s interval, and 3 s or 5 s working time. The purified DNA fragments were enriched, and their concentrations and purity were detected. Polymerase chain reaction （PCR） and agarose eleetrophoresis were performed to check the results. Results The appropriate ChIP experimental conditions in prostate cancer cells were as follows ： （ 1 ） working time of 3 s every 38 s or 5 s every 55 s with a 5 mm mierotip at a power setting of 30% of the maximum power, in total time from 45 s to 75 s; （2） If the concentration of purified DNA could be in more than 10 ng/μl or purity value （A260/A280） in 1.45 above, we could get a better result. Conclusion In prostate cancer cells （PC -3, and DU145） , when we selected the appropriate ultrasonic frequency, the general ultrasonic working time was about 60 s and the right DNA fragments could be obtained. Fixing volume of elution liquid, it was worth centrifugal twice than one to obtain a higher DNA purity value. What＇ s more, the higher purity of DNA, the more obvious of results we could arrive at.