Abstract
Multiple analyses such as DNA profiling, sequencing, or comparative genome hybridization (CGH) done on the single-cell level long for pre-amplification due to the diploid human genome. Isothermal whole genome amplification allows amplification of long DNA templates from single cells. When analysis needs to be performed under rare cell conditions additional care needs to be taken due to the fact that, even after pre-enrichment, few candidate target cells are still dispersed among an overwhelming number of non-target background cells. Here, we describe a protocol where we define a population of candidate target cells based on specific staining. Candidate cells are then isolated by laser microdissection and pressure catapulting (LMPC) and transferred onto a microliter reaction slide. This slide allows monitoring the single-cell isolation process and isothermal whole genome amplification in less than 2 μL. The amplification products obtained from single cells can be forwarded to multiple analyses.
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Acknowledgement
This work was supported by the EU SAFE Network of Excellence (LSHB-CT-2004-503243, EU 6th Framework Package) and the County of Styria, Austria.
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Kroneis, T., Chen, S., El-Heliebi, A. (2015). Low-Volume On-Chip Single-Cell Whole Genome Amplification for Multiple Subsequent Analyses. In: Kroneis, T. (eds) Whole Genome Amplification. Methods in Molecular Biology, vol 1347. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-2990-0_17
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DOI: https://doi.org/10.1007/978-1-4939-2990-0_17
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