Abstract
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.
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Acknowledgments
This Research was supported (in part) by the Intramural Research Program of the NIH, National Cancer Institute, Center for Cancer Research. This project has been funded in whole or in part with federal funds from the National Cancer Institute, National Institutes of Health, under Contract No. HHSN261200800001E. The content of this publication does not necessarily reflect the views or policies of the Department of Health and Human Services, nor does mention of trade names, commercial products, or organizations imply endorsement by the U.S. Government.
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Castro, N.P., Golubeva, Y.G. (2018). Adaptation of Laser Microdissection Technique to Nanostring RNA Analysis in the Study of a Spontaneous Metastatic Mammary Carcinoma Mouse Model. In: Murray, G. (eds) Laser Capture Microdissection. Methods in Molecular Biology, vol 1723. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-7558-7_6
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DOI: https://doi.org/10.1007/978-1-4939-7558-7_6
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