Castro Nadia P, Golubeva Yelena G
Tumor Growth Factor Section, Mouse Cancer Genetics Program, National Cancer Institute, Frederick, MD, USA.
Cancer Genomic Research Laboratory (CGR), Division of Cancer Epidemiology and Genetics, NCI, FNLCR, Leidos Biomedical Research, Inc, Gaithersburg, MD, USA.
Methods Mol Biol. 2018;1723:119-137. doi: 10.1007/978-1-4939-7558-7_6.
The mouse model characterized by spontaneous lung metastasis from JygMC (A) cells closely resembles the human triple negative breast cancer (TNBC) subtype. The primary tumors morphologically present both epithelial and spindle-like cells, but metastases in lung parenchyma display only adenocarcinoma properties. In the study of molecular signatures, laser capture microdissection (LCM) on frozen tissue sections was used to separate the following regions of interest: the epithelial-mesenchymal transition (EMT), mesenchymal-epithelial transition (MET), carcinoma, lung metastases, normal mammary gland and normal lung parenchyma. NanoString was selected for the study of molecular signatures in LCM targets as a reliable downstream gene expression platform allowing analysis of tissue lysates without RNA extraction and amplification. This chapter provides detailed protocols for the collection of tissue, LCM sample preparation and dissection, production of lysates, extraction, and quality control of RNA for NanoString analysis, as well as the methodology of Nanostring gene expression profiling experiment.
以JygMC (A)细胞自发肺转移为特征的小鼠模型与人类三阴性乳腺癌(TNBC)亚型极为相似。原发性肿瘤在形态上同时呈现上皮样细胞和梭形细胞，但肺实质中的转移灶仅表现出腺癌特征。在分子特征研究中，利用冷冻组织切片上的激光捕获显微切割(LCM)技术分离出以下感兴趣的区域：上皮-间质转化(EMT)、间质-上皮转化(MET)、癌组织、肺转移灶、正常乳腺组织和正常肺实质。选择NanoString作为LCM靶点分子特征研究的可靠下游基因表达平台，该平台无需RNA提取和扩增即可分析组织裂解液。本章详细介绍了组织收集、LCM样本制备与切割、裂解液制备、RNA提取及用于NanoString分析的RNA质量控制的实验方案，以及NanoString基因表达谱实验方法。
