激光捕获显微切割结合RNA线性扩增技术在膀胱移行细胞癌相关基因研究中的应用

Application of laser capture microdissection combined with RNA In vitro linear amplification in the research of the relevant genes of bladder cancer

目的当对有间质细胞混杂的膀胱移行上皮细胞与癌细胞进行基因差异表达分析时,激光捕获显微切割技术(LCM)是不可缺少的。LCM与RNA线性扩增结合,目的是确定一条可行的技术路线,获取均质的、足量的RNA,以备进一步用于膀胱癌相关基因研究。方法采用LCM技术分别从正常膀胱黏膜及膀胱癌组织冰冻切片中获取膀胱移行上皮细胞及癌细胞,提取RNA,并对120ng上皮细胞RNA进行线性扩增,获得aRNA20μg。用逆转录聚合酶链反应(RTPCR)验证扩增前、后RNA中βactin基因表达水平。结果对照实验Ⅰ证实LCM后RNA完整性较好;产物RNA扩增后获得片段大小为0.5～2.5kb的aRNA,且βactin表达完整。结论LCM结合RNA线性扩增技术获取均一的、足量的、完整性好的目的细胞RNA,能用于进一步研究。

Objective To determine a feasible technical routine combining laser capture microdisection (LCM) with RNA linear amplification in vitro to obtain pure,sufficient,integrative RNA for the using in further research on the relevant genes of bladder transitional cell carcinoma (BTCC).Methods Bladder transitional cells were obtained from frozen bladder membrane sections and BTCC cells from frozen BTCC tissue sections by using LCM.RNA was extracted and about 120 ng RNA was linearly amplified in vitro,then 20 ug aRNA was obtained.The expression levels of β-actin in primary total RNA and amplified RNA were detected by using RT-PCR.Results[WT5”BZ] RNA integrity is good after LCM confirmed by contral experimentⅠ;About 0.5～ 2.5 kb RNA fragments were obtained after RNA amplification and β-actin expression was integral.Conclusion LCM combined with RNA pinear amplification in vitro can be apllied to obtain pure,sufficient,integrative RNA for the using in further research.