title: A robust method to analyze copy number alterations of less than 100 kb in single cells using oligonucleotide array CGH.
abstract: Comprehensive genome wide analyses of single cells became increasingly important in cancer research, but remain to be a technically challenging task. Here, we provide a protocol for array comparative genomic hybridization (aCGH) of single cells. The protocol is based on an established adapter-linker PCR (WGAM) and allowed us to detect copy number alterations as small as 56 kb in single cells. In addition we report on factors influencing the success of single cell aCGH downstream of the amplification method, including the characteristics of the reference DNA, the labeling technique, the amount of input DNA, reamplification, the aCGH resolution, and data analysis. In comparison with two other commercially available non-linear single cell amplification methods, WGAM showed a very good performance in aCGH experiments. Finally, we demonstrate that cancer cells that were processed and identified by the CellSearch® System and that were subsequently isolated from the CellSearch® cartridge as single cells by fluorescence activated cell sorting (FACS) could be successfully analyzed using our WGAM-aCGH protocol. We believe that even in the era of next-generation sequencing, our single cell aCGH protocol will be a useful and (cost-) effective approach to study copy number alterations in single cells at resolution comparable to those reported currently for single cell digital karyotyping based on next generation sequencing data.
authors.affiliation: Department of Surgery (A), Heinrich-Heine University and University Hospital Düsseldorf, Düsseldorf, Germany. birte.moehlendick@uni-duesseldorf.de
authors.forename: Birte
authors.initials: B
authors.lastname: Möhlendick
authors.forename: Christoph
authors.initials: C
authors.lastname: Bartenhagen
authors.forename: Bianca
authors.initials: B
authors.lastname: Behrens
authors.forename: Ellen
authors.initials: E
authors.lastname: Honisch
authors.forename: Katharina
authors.initials: K
authors.lastname: Raba
authors.forename: Wolfram T
authors.initials: WT
authors.lastname: Knoefel
authors.forename: Nikolas H
authors.initials: NH
authors.lastname: Stoecklein
mesh_terms: D020029:Base Pairing; D055028:Comparative Genomic Hybridization / Q000191:economics / Q000379:methods*; D003362:Cost-Benefit Analysis; D056915:DNA Copy Number Variations / Q000235:genetics*; D005260:Female; D006801:Humans; D008297:Male; D020411:Oligonucleotide Array Sequence Analysis / Q000191:economics / Q000379:methods*; D016133:Polymerase Chain Reaction; D059010:Single-Cell Analysis / Q000191:economics / Q000379:methods*