Abstract
There is an increasing requirement for genetic analysis of individual cells from tissue sections. This is particularly the case for analysis of tumor cells but is also a requirement for analysis of cells in pancreas from individuals with type 1 diabetes where there is evidence of viral infection or in the analysis of chimerism in pancreas; either post-transplant or as a result of feto-maternal cell transfer.
This protocol describes a strategy to isolate cells using laser microdissection and to run a 17plex PCR to discriminate between cells of haplo-identical origin (i.e., fetal and maternal cells) in pancreas tissue but other robust DNA tests could be used. In short, snap-frozen tissues are cryo-sectioned and mounted onto membrane-coated slides. Target cells are harvested from the tissue sections by laser microdissection and pressure catapulting (LMPC) prior to DNA profiling. This is based on amplification of highly repetitive yet stably inherited loci (short tandem repeats, STR) as well as the amelogenin locus for sex determination and separation of PCR products by capillary electrophoresis.
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Acknowledgement
This work was supported by EU SAFE Network of Excellence (LSHB-CT-2004-503243, EU 6th Framework Package) and the County of Styria, Austria as well as a Diabetes UK grant to KMG (ref 12/0004564). We are grateful to nPOD (http://www.jdrfnpod.org/) for supplying pancreas tissues presectioned on membrane slides for these experiments and to Dr Peter Sedlmayr, Institute of Cell Biology, Medical University of Graz for facilitating the protocol development in his laboratory.
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Kroneis, T., Ye, J., Gillespie, K. (2015). Laser Capture and Single Cell Genotyping from Frozen Tissue Sections. In: Gillespie, K. (eds) Type-1 Diabetes. Methods in Molecular Biology, vol 1433. Humana Press, New York, NY. https://doi.org/10.1007/7651_2015_290
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DOI: https://doi.org/10.1007/7651_2015_290
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Publisher Name: Humana Press, New York, NY
Print ISBN: 978-1-4939-3641-0
Online ISBN: 978-1-4939-3643-4
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