摘要
背景与目的 :激光捕获显微切割技术 (LCM)是获取均一目的细胞的有效方法。利用LCM技术从膀胱粘膜中分离膀胱移行上皮细胞从肿瘤间质细胞中分离膀胱癌细胞 ,进行RNA提取、纯化、浓缩以备进一步研究。方法 :采用LCM技术分别从正常膀胱粘膜及膀胱癌组织冰冻切片中获取膀胱移行上皮细胞及膀胱癌细胞 ,提取RNA ,并对微量RNA进行纯化、浓缩。然后用RT PCR验证TotalRNA中β actin基因表达水平。结果 :对照实验Ⅰ证实经LCM后RNA完整性较好 ;经对照实验Ⅱ初步确定设定条件下LCMshooting次数与可获得RNA量间对应关系。从膀胱粘膜中捕获膀胱移行上皮细胞 2 5万shootings;从膀胱癌组织中获取癌细胞 2 0万shootings。经RT PCR验证β actin基因表达表达完整。结论 :使用LCM技术能成功地获取较为均一的研究目的细胞 ,RNA完整性较好 ,能用于进一步研究中。
Objective:Laser Capture Microdisection (LCM) has become indispensable for obtaining the homogeneous objective cells.Transitional cells were isolated from bladder membrane and carcinoma cells were separated from stromal cells by LCM.RNA were extracted,purified and concentrated from laser capture microdissected cells for further use.Methods: Bladder transitional cells were obtained from bladder tissue,bladder transitional cell carcinoma (BTCC) cells from BTCC tissue by frozen sections and LCM.Then RNA was extracted,purified and concentrated.The expression level of β actin in total RNA was detected by RT PCR.Results:RNA integrity is good after LCM confirmed by control experimentⅠ;By control experiment Ⅱ,the correlation between the number of LCM shooting and RNA quantity under arranged conditions was preliminary conformed.β actin level was integral.Conclusion:LCM can be applied to successfully obtain purely objective cells for research.The integrity of RNA is good and can be used in further research.
出处
《中国生物工程杂志》
CAS
CSCD
北大核心
2005年第3期74-78,共5页
China Biotechnology
