Hu Sheng-ping, Zhang Ke-hao, Shen Zhong-ying, Wu Ming-yao, Deng Xiao-ling, Yang Jie-sheng, Fang Fu-de
Center for Molecular Biology, Shantou University Medical College, Shantou 515031, China.
Zhongguo Yi Xue Ke Xue Yuan Xue Bao. 2003 Dec;25(6):659-63.
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.
Blocks of EC were stored at -70 degrees C as close as possible to the time of surgical resection. The tissue block was embedded in OCT and frozen sections of 35 microns in thickness were cut in a cryostat under strict RNAse-free conditions. Individual frozen sections were mounted on plain glass slides and 30-gauge needle attached to a 1 ml syringe was used to microdissect defined cells in the sections. The procured cells were used for total RNA extraction.
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.
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.
从异质性组织块中分离出感兴趣的细胞，以获得肿瘤细胞所获得分子改变的准确表征，从而满足对食管癌（EC）进展过程中基因表达模式进一步研究的需求。
尽可能在手术切除后立即将EC组织块保存在-70℃。将组织块包埋于OCT中，在严格无RNA酶条件下，用冷冻切片机切成35微米厚的冰冻切片。将单个冰冻切片置于普通载玻片上，用连接1ml注射器的30号针头在切片中显微切割特定细胞。所获得的细胞用于总RNA提取。
成功开发了一种优化的手动显微切割方案，可精确切割面积小至1/25mm2的区域。通过琼脂糖凝胶电泳评估18S和28S rRNA，从所获得细胞中回收的RNA质量高，适合后续分子分析应用。
据信，手动显微切割能够从复杂的原始组织中获取特定的细胞群体，从而能够研究组织、细胞和功能特异性的基因表达模式。该技术简单、易于操作、用途广泛，在实际无法使用激光捕获显微切割（LCM）时特别有用。
