title: Combined molecular genetic and cytogenetic analysis from single cells after isothermal whole-genome amplification.
abstract: BACKGROUND
  Analysis of chromosomal aberrations or single-gene disorders from rare fetal cells circulating in the blood of pregnant women requires verification of the cells' genomic identity. We have developed a method enabling multiple analyses at the single-cell level that combines verification of the genomic identity of microchimeric cells with molecular genetic and cytogenetic diagnosis.
  
  
  METHODS
  We used a model system of peripheral blood mononuclear cells spiked with a colon adenocarcinoma cell line and immunofluorescence staining for cytokeratin in combination with DNA staining with the nuclear dye TO-PRO-3 in a preliminary study to define candidate microchimeric (tumor) cells in Cytospin preparations. After laser microdissection, we performed low-volume on-chip isothermal whole-genome amplification (iWGA) of single and pooled cells.
  
  
  RESULTS
  DNA fingerprint analysis of iWGA aliquots permitted successful identification of all analyzed candidate microchimeric cell preparations (6 samples of pooled cells, 7 samples of single cells). Sequencing of 3 single-nucleotide polymorphisms was successful at the single-cell level for 20 of 32 allelic loci. Metaphase comparative genomic hybridization (mCGH) with iWGA products of single cells showed the gains and losses known to be present in the genomic DNA of the target cells.
  
  
  CONCLUSIONS
  This method may be instrumental in cell-based noninvasive prenatal diagnosis. Furthermore, the possibility to perform mCGH with amplified DNA from single cells offers a perspective for the analysis of nonmicrochimeric rare cells exhibiting genomic alterations, such as circulating tumor cells.
authors.affiliation: Institute of Cell Biology, Histology and Embryology, Center for Molecular Medicine, Medical University Graz, Graz, Austria.
authors.forename: Thomas
authors.initials: T
authors.lastname: Kroneis
authors.forename: Jochen B
authors.initials: JB
authors.lastname: Geigl
authors.forename: Amin
authors.initials: A
authors.lastname: El-Heliebi
authors.forename: Martina
authors.initials: M
authors.lastname: Auer
authors.forename: Peter
authors.initials: P
authors.lastname: Ulz
authors.forename: Thomas
authors.initials: T
authors.lastname: Schwarzbraun
authors.forename: Gottfried
authors.initials: G
authors.lastname: Dohr
authors.forename: Peter
authors.initials: P
authors.lastname: Sedlmayr
mesh_terms: D046528:Chimerism; D002869:Chromosome Aberrations; D055028:Comparative Genomic Hybridization / Q000379:methods; D020732:Cytogenetic Analysis / Q000379:methods*; D016172:DNA Fingerprinting / Q000379:methods*; D005260:Female; D005455:Fluorescent Antibody Technique; D015894:Genome, Human; D019073:HT29 Cells; D006801:Humans; D007633:Keratins / Q000378:metabolism; D007963:Leukocytes, Mononuclear / Q000166:cytology* / Q000378:metabolism*; D008677:Metaphase; D009360:Neoplastic Cells, Circulating / Q000378:metabolism / Q000473:pathology; D021141:Nucleic Acid Amplification Techniques; D011247:Pregnancy; D011296:Prenatal Diagnosis / Q000379:methods; D017422:Sequence Analysis, DNA / Q000379:methods; D059010:Single-Cell Analysis / Q000379:methods*
chemical_list: D007633:Keratins