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Published on: November 26, 2015
[Manual microdissection of defined cells and RNA extraction for gene expression analysis of esophageal carcinoma
Sheng-ping Hu1, Ke-hao Zhang, Zhong-ying Shen
1Center for Molecular Biology, Shantou University Medical College, Shantou 515031, China. sphu@stu.edu.cn
Objectives:
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 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.
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.
