Open AccessAutomatic retrieval of single microchimeric cells and verification of identity by on‐chip multiplex PCR
Author(s) -
Kroneis Thomas,
GutsteinAbo Liat,
Kofler Kristina,
Hartmann Michaele,
Hartmann Petra,
AlunniFabbroni Marianna,
Walcher Wolfgang,
Dohr Gottfried,
Petek Erwin,
Guetta Esther,
Sedlmayr Peter
Publication year - 2010
Publication title -
journal of cellular and molecular medicine
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.44
H-Index - 130
eISSN - 1582-4934
pISSN - 1582-1838
DOI - 10.1111/j.1582-4934.2009.00784.x
Subject(s) - multiplex , dna profiling , biology , microchimerism , laser capture microdissection , immunofluorescence , microbiology and biotechnology , computational biology , genetics , dna , antibody , fetus , gene , pregnancy , gene expression
The analysis of rare cells is not an easy task. This is especially true when cells representing a fetal microchimerism are to be utilized for the purpose of non‐invasive prenatal diagnosis because it is both imperative and difficult to avoid contaminating the minority of fetal cells with maternal ones. Under these conditions, even highly specific biochemical markers are not perfectly reliable. We have developed a method to verify the genomic identity of rare cells that combines automatic screening for enriched target cells (based on immunofluorescence labelling) with isolation of single candidate microchimeric cells (by laser microdissection and subsequent laser catapulting) and low‐volume on‐chip multiplex PCR for DNA fingerprint analysis. The power of the method was tested using samples containing mixed cells of related and non‐related individuals. Single‐cell DNA fingerprinting was successful in 74% of the cells analysed (55/74), with a PCR efficiency of 59.2% (860/1452) for heterozygous loci. The identification of cells by means of DNA profiling was achieved in 100% (12/12) of non‐related cells in artificial mixtures and in 86% (37/43) of cells sharing a haploid set of chromosomes and was performed on cells enriched from blood and cells isolated from tissue. We suggest DNA profiling as a standard for the identification of microchimerism on a single‐cell basis.