title: Low-Volume On-Chip Single-Cell Whole Genome Amplification for Multiple Subsequent Analyses.
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.
authors.affiliation: Research Unit for Single Cell Analysis, Institute of Cell Biology, Histology and Embryology, Medical University of Graz, Harrachgasse 21, Graz, 8010, Austria. thomas.kroneis@medunigraz.at.|Sahlgrenska Cancer Center, University of Gothenburg, Gothenburg, Sweden. thomas.kroneis@medunigraz.at.
authors.forename: Thomas
authors.initials: T
authors.lastname: Kroneis
authors.affiliation: Research Unit for Single Cell Analysis, Institute of Cell Biology, Histology and Embryology, Medical University of Graz, Harrachgasse 21, Graz, 8010, Austria.
authors.forename: Shukun
authors.initials: S
authors.lastname: Chen
authors.affiliation: Research Unit for Single Cell Analysis, Institute of Cell Biology, Histology and Embryology, Medical University of Graz, Harrachgasse 21, Graz, 8010, Austria.
authors.forename: Amin
authors.initials: A
authors.lastname: El-Heliebi
mesh_terms: D016678:Genome; D023281:Genomics / Q000379:methods*; D007150:Immunohistochemistry; D061346:Laser Capture Microdissection; D046210:Microfluidic Analytical Techniques; D021141:Nucleic Acid Amplification Techniques; D059010:Single-Cell Analysis / Q000379:methods*
keywords: AmpliGrid; Isothermal whole genome amplification; Laser microdissection; Single-cell analysis