改良手工显微切割法获取特定细胞提取高质量RNA

Modified Manual Microdissection in Procuring Defined Cells for High-Qulitity RNA Acquisition

探讨显微切割过程中有效保持RNA完整性的组织固定方法,建立一种简易的手工显微切割法.应用自制“T形板”辅助冰冻切片,100%无水乙醇一次性脱水固定,“排除切割法”获取目的细胞,用TRIzol提取RNA,琼脂糖凝胶电泳和RT-PCR分析RNA质量.“一步法”固定可长时间保存RNA的完整性;从食管癌标本5个特定阶段的细胞中提取的RNA,经电泳和RT-PCR分析均具有较高的质量.无水乙醇“一步法”固定,在显微切割的过程中可有效保持RNA的完整性;T形板和“排除切割法”简化了手工显微切割的操作,提取的RNA质、量均可满足后续分子水平研究的需要.

An optimal fixation protocol was determined to stabilize RNA during microdissection and obtain high-quality RNA from specific cell populations procured from esophageal carcinoma specimens. A self-made T-shape plate （T plate）, one-step dehydration of tissue sections in 100% ethanol immediately following cryosectioning and ＂exclusion microdissection＂ were used to develop an effective manual microdissection procedure. The RNA quality isolated from dissected cells was analyzed by neutral agarose gel electrophoresis and reverse transcription-polymerase chain reaction （RT-PCR）. One-step 100% ethanol fixation of cryosections effectively stabilized RNA integrity for agelong period of time while maintaining histological morphology comparable to that using the conventional procedure. The RNA isolated from 5 different stagespecific cell populations of esophageal carcinoma specimens was of high quality and sufficient quantity for various downstream molecular analyses. In conjunction with the application of the T plate and ＂exclusion microdissection＂ procedure, one-step 100% ethanol fixation can efficiently simplify manual microdissection procedure. The method is suitable for a wide spectrum of expression analysis in diverse clinical settings.