title: Analysis of Copy-Number Alterations in Single Cells Using Microarray-Based Comparative Genomic Hybridization (aCGH).
abstract: In this unit, we describe a workflow that enables array comparative genomic hybridization (aCGH) of single cells. The unit first describes the isolation and preparation of single peripheral mononuclear cells from blood (PBMC) to prepare a suitable reference DNA for aCGH experiments. An alternative procedure is described for the preparation of single cells of GM14667 and GM05423 cell lines to use as reference DNA and for sex-mismatched control experiments. A guide is also provided for micromanipulation of single cells. Next, the unit describes whole-genome amplification using adapter-linker PCR (Ampli1 WGA Kit) and an alternative nonlinear WGA method (PicoPLEX WGA Kit) for single-cell amplification. A protocol is also included for reamplification of Ampli1 WGA products, which can be used for aCGH as well. Finally, the use of 4 × 180k oligonucleotide microarrays to perform aCGH with single-cell WGA products is described.
authors.affiliation: Department of Surgery (A), Heinrich Heine University and University Hospital Düsseldorf, Düsseldorf, Germany.
authors.forename: Birte
authors.initials: B
authors.lastname: Möhlendick
authors.forename: Nikolas H
authors.initials: NH
authors.lastname: Stoecklein
mesh_terms: D000818:Animals; D002460:Cell Line; D055028:Comparative Genomic Hybridization / Q000295:instrumentation / Q000379:methods*; D018628:Gene Dosage; D006801:Humans; D007963:Leukocytes, Mononuclear; D020411:Oligonucleotide Array Sequence Analysis / Q000295:instrumentation / Q000379:methods*
keywords: aCGH; adapter-linker-PCR; copy-number alterations; single-cell analysis; whole genome amplification