摘要
目的建立手工组织显微切割方法准确挖取食管癌不同病变阶段细胞,从中提取高质量RNA,以满足后续基因表达分析的需要。方法常规制备冰冻组织切片,用无水乙醇一次性脱水固定,显微镜下用1ml注射器针头从组织冰冻切片中分别挖取不同病变阶段的细胞,用TRIzol提取总RNA。结果手工挖取面积可准确到1/25mm2,从挖取的细胞中提取出高质量的总RNA,凝胶电泳分析见清晰的18S和28SrRNA带,可满足后续分子水平研究的要求。结论无水乙醇固定冰冻组织切片可有效保存RNA的完整性,手工组织显微切割能够准确挖取小至1/25mm2面积的细胞,方法简单、易掌握、易操作,不须特殊仪器设备,适合在一般实验室推广应用。
To isolate cells of interest from heterogeneous tissue blocks to obtain accurate representations of molecular alterations acquired by neoplastic cells so as to meet the demands of further study on gene expression patterns of the esophageal carcinoma(EC)evolution. Methods Blocks of EC were stored at -70℃ as close as possible to the time of surgical resection. The tissue block was embedded in OCT and frozen sections of 35 μm in thickness were cut in a cryostat under strictRNAse-free conditions. Individual frozen sections were mounted on plain glass slides and 30-gauge needle attached to a 1ml syringe was used to microdissect defined cells in the sections. The procured cells were used for total RNA extraction. Results An optimized protocol of manual microdissection was developed successfully whereby regions with an area as small as 1/25 mm2 could be accurately dissected. The RNA recovered from procured cells was of high quality suitable for subsequent applications of molecular analysis as assessed of 18S and 28S rRNAs by electrophoresis on agarose gel. Conclusions It is believed that manual microdissection is capable to procure defined cell populations from complex primary tissues, thus allowing investigation of tissue-, cell-, and function-specific gene expression patterns. The technique is simple, easy to perform, versatile, and of particular usefulness when laser capture microdissection(LCM)is practically unavailable.
出处
《中国医学科学院学报》
CAS
CSCD
北大核心
2003年第6期659-663,共5页
Acta Academiae Medicinae Sinicae
基金
广东省自然科学基金(000819)
国家教育部留学回国人员科研启动基金(外教司2001345号)
汕头大学研究与发展基金(L00007)资助~~
关键词
手工组织显微切割
食管癌
RNA提取
manual tissue microdissection
esophageal carcinoma
RNA extractionActa Acad Med Sin, 2003,25(6)659 ～ 663
