title: Single-cell PCR of genomic DNA enabled by automated single-cell printing for cell isolation.
abstract: Single-cell analysis has developed into a key topic in cell biology with future applications in personalized medicine, tumor identification as well as tumor discovery (Editorial, 2013). Here we employ inkjet-like printing to isolate individual living single human B cells (Raji cell line) and load them directly into standard PCR tubes. Single cells are optically detected in the nozzle of the microfluidic piezoelectric dispenser chip to ensure printing of droplets with single cells only. The printing process has been characterized by using microbeads (10µm diameter) resulting in a single bead delivery in 27 out of 28 cases and relative positional precision of ±350µm at a printing distance of 6mm between nozzle and tube lid. Process-integrated optical imaging enabled to identify the printing failure as void droplet and to exclude it from downstream processing. PCR of truly single-cell DNA was performed without pre-amplification directly from single Raji cells with 33% success rate (N=197) and Cq values of 36.3±2.5. Additionally single cell whole genome amplification (WGA) was employed to pre-amplify the single-cell DNA by a factor of >1000. This facilitated subsequent PCR for the same gene yielding a success rate of 64% (N=33) which will allow more sophisticated downstream analysis like sequencing, electrophoresis or multiplexing.
authors.affiliation: HSG-IMIT - Institut für Mikro- und Informationstechnik, Georges-Koehler-Allee 103, 79110 Freiburg, Germany. Electronic address: Fabian.stumpf@hsg-imit.de.
authors.forename: F
authors.initials: F
authors.lastname: Stumpf
authors.affiliation: Laboratory for MEMS Applications, Department of Microsystems Engineering - IMTEK, University of Freiburg, Georges-Koehler-Allee 103, 79110 Freiburg, Germany; Cytena GmbH - University of Freiburg, Georges-Koehler-Allee 103, 79110 Freiburg, Germany. Electronic address: jonas.schoendube@imtek.de.
authors.forename: J
authors.initials: J
authors.lastname: Schoendube
authors.affiliation: Laboratory for MEMS Applications, Department of Microsystems Engineering - IMTEK, University of Freiburg, Georges-Koehler-Allee 103, 79110 Freiburg, Germany; Cytena GmbH - University of Freiburg, Georges-Koehler-Allee 103, 79110 Freiburg, Germany. Electronic address: andre.gross@imtek.de.
authors.forename: A
authors.initials: A
authors.lastname: Gross
authors.affiliation: Laboratory for Microarray Copying, Centre for Biological Systems Analysis (ZBSA), University of Freiburg, 79104 Freiburg, Germany. Electronic address: christin.rath@zbsa.uni-freiburg.de.
authors.forename: C
authors.initials: C
authors.lastname: Rath
authors.affiliation: Laboratory for MEMS Applications, Department of Microsystems Engineering - IMTEK, University of Freiburg, Georges-Koehler-Allee 103, 79110 Freiburg, Germany. Electronic address: sonja.niekrawietz@imtek.de.
authors.forename: S
authors.initials: S
authors.lastname: Niekrawietz
authors.affiliation: Laboratory for MEMS Applications, Department of Microsystems Engineering - IMTEK, University of Freiburg, Georges-Koehler-Allee 103, 79110 Freiburg, Germany. Electronic address: peter.koltay@imtek.de.
authors.forename: P
authors.initials: P
authors.lastname: Koltay
authors.affiliation: BIOSS - Centre for Biological Signalling Studies, University of Freiburg, 79110 Freiburg, Germany; Laboratory for Microarray Copying, Centre for Biological Systems Analysis (ZBSA), University of Freiburg, 79104 Freiburg, Germany. Electronic address: guenter.roth@zbsa.uni-freiburg.de.
authors.forename: G
authors.initials: G
authors.lastname: Roth
mesh_terms: D001802:Blood Substitutes; D045744:Cell Line, Tumor; D002874:Chromosome Mapping / Q000295:instrumentation*; D016287:Computer Peripherals; D004247:DNA / Q000235:genetics*; D004867:Equipment Design; D019544:Equipment Failure Analysis; D006801:Humans; D056656:Lab-On-A-Chip Devices; D008223:Lymphoma / Q000473:pathology*; D021141:Nucleic Acid Amplification Techniques / Q000295:instrumentation; D016133:Polymerase Chain Reaction / Q000295:instrumentation*; D012371:Robotics / Q000295:instrumentation
chemical_list: D001802:Blood Substitutes; D004247:DNA
keywords: Label free; Optical cell detection; Piezoelectric printing; Single-cell PCR; Single-cell printing; Whole genome amplification