Yang Yan-Qing, Zhang Wen, Zhang Bao-Feng, Gao Heng-Jun, Zhang Qing-Hua
State Key Laboratory of Medical Genomics and Shanghai Institute of Hematology, Ruijin Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai 200025, China.
Yi Chuan. 2008 Nov;30(11):1521-6. doi: 10.3724/sp.j.1005.2008.01521.
We developed a standard protocol for quality assessment of low amount RNA from the cells obtained by laser capture microdissection (LCM). Three gastric noncancerous tissues were cryo-sectioned, stained with Cresyl Violet, and pathologically rechecked. Epithelial cells were obtained by LCM and RNA was isolated. Agilent 2100 bioanalyzer was used to check the RNA quality. To validate the results from 2100 bioanalyzer, RT-PCR was performed with six genes at both 5'and 3' end-regions of different abundance (EF1A and ATCB of high abundance, GAPDH and B2M of moderate abundance, and MED1 and CK20 of low abundance). RT-PCR analysis of 3 good quality RNAs from cultured cell lines and 3 poor quality RNAs from gastric noncancerous tissues showed high correlations with that from 2100 bioanalyzer. In conclusion, the pipeline for low amount RNA quality assessment by RT-PCR from tissue cryo-section, pathological recheck, LCM purification and RNA isolation is applicable as a routine method in cancer genome research.
我们开发了一种用于评估通过激光捕获显微切割(LCM)获得的细胞中低量RNA质量的标准方案。对三个胃非癌组织进行冷冻切片,用甲酚紫染色,并进行病理复查。通过LCM获取上皮细胞并分离RNA。使用安捷伦2100生物分析仪检查RNA质量。为了验证2100生物分析仪的结果,对不同丰度的六个基因在5'和3'末端区域进行了逆转录聚合酶链反应(RT-PCR)(高丰度的EF1A和ATCB、中等丰度的GAPDH和B2M,以及低丰度的MED1和CK20)。对来自培养细胞系的3个高质量RNA和来自胃非癌组织的3个低质量RNA进行的RT-PCR分析与2100生物分析仪的分析结果高度相关。总之,通过RT-PCR从组织冷冻切片、病理复查、LCM纯化和RNA分离来评估低量RNA质量的流程可作为癌症基因组研究中的常规方法应用。