摘要
目的：为寻找一种操作简便、结果稳定可靠的组织处理及切片染色技术，尝试应用组织切片手工显微切割后快速提取DNA的方法，观察提取的DNA质、量是否能满足后续分子水平研究的需要。方法：实验于2004-07／2007-07在广东医学院病理学实验室完成。取广东医学院附属医院病理科提供的子宫颈癌石蜡包埋组织切片，将切片贴于载玻片长时间完全脱蜡后，用苏木精对细胞核进行淡染，再在倒转置显微镜下进行显微切割，将切割下的组织放入装有提取缓冲液的EP管中，整个过程不需更换EP管，不经过复杂的酚-氯仿抽提，采用细胞裂解并蛋白酶K消化的方法，快速、简便提取DNA。结果：经分光光度计检测提取的DNA浓度为0．14～5．25g／L，A260/A280值在1．6～1．8；PCR检测扩增出了预期长度为231bp的目的片段。结论：手工组织显微切割后快速DNA提取法可以提供足量浓度的DNA，PCR反应模板结果稳定可靠，可满足后续分子水平研究的需要。
AIM： To explore a simple, reliable method for tissue processing and section staining by extracting DNA from the manually microdissectioned specimen, and to identify whether the extracted DNA is useful in the following study at molecule level. METHODS： The experiment was performed at the pathological laboratory of Guangdong Medical College from July 2004 to July 2007. The paraffin imbedding tissue sections of cervical cancer were thoroughly deparaffinized after mounted on slides for a long period of time. The nucleus was slightly stained with hematoxylin and microdissectioned under inverted microscope. The microdissectioned samples were put into EP tubes filled with digestion buffer to split the cells and then the DNA was extracted. During the whole course, PE tubes did not change, and the complicated phenol/chloroform extraction did not perform. The DNA extraction was rapid and simple. RESULTS： The DNA was measured by the spectrophotometer with concentrations from 0.14 to 5.25 g/L and absorbance values of A260/A280 were 1.6-1.8. All samples were amplified with PCR to produce expected length specific target fragment （231 bp）. CONCLUSION： Rapid DNA extraction after manual tissue microdissection can produce adequate amount of DNA and maintain good quality of DNA template for PCR. The DNA meets the need of the following molecular experiments.
出处
《中国组织工程研究与临床康复》
CAS
CSCD
北大核心
2008年第24期4677-4679,共3页
Journal of Clinical Rehabilitative Tissue Engineering Research
基金
湛江市科技招标项目(ZZ0405)~~
