title: Quantification of cell-free DNA in normal and complicated pregnancies: overcoming biological and technical issues.
abstract: The characterization of cell-free DNA (cfDNA) originating from placental trophoblast in maternal plasma provides a powerful tool for non-invasive diagnosis of fetal and obstetrical complications. Due to its placental origin, the specific epigenetic features of this DNA (commonly known as cell-free fetal DNA) can be utilized in creating universal 'fetal' markers in maternal plasma, thus overcoming the limitations of gender- or rhesus-specific ones. The goal of this study was to compare the performance of relevant approaches and assays evaluating the amount of cfDNA in maternal plasma throughout gestation (7.2-39.5 weeks). Two fetal- or placental-specific duplex assays (RPP30/SRY and RASSF1A/β-Actin) were applied using two technologies, real-time quantitative PCR (qPCR) and droplet digital PCR (ddPCR). Both methods revealed similar performance parameters within the studied dynamic range. Data obtained using qPCR and ddPCR for these assays were positively correlated (total cfDNA (RPP30): R = 0.57, p = 0.001/placental cfDNA (SRY): R = 0.85, p<0.0001; placental cfDNA (RASSF1A): R = 0.75, p<0.0001). There was a significant correlation in SRY and RASSF1A results measured with qPCR (R = 0.68, p = 0.013) and ddPCR (R = 0.56, p = 0.039). Different approaches also gave comparable results with regard to the correlation of the placental cfDNA concentration with gestational age and pathological outcome. We conclude that ddPCR is a practical approach, adaptable to existing qPCR assays and well suited for analysis of cell-free DNA in plasma. However, it may need further optimization to surpass the performance of qPCR.
authors.affiliation: Department of Medical Genetics, University of British Columbia, Vancouver, British Columbia, Canada; Child & Family Research Institute, Vancouver, British Columbia, Canada.
authors.forename: Irina
authors.initials: I
authors.lastname: Manokhina
authors.affiliation: Department of Medical Genetics, University of British Columbia, Vancouver, British Columbia, Canada; Child & Family Research Institute, Vancouver, British Columbia, Canada.
authors.forename: Tanjot K
authors.initials: TK
authors.lastname: Singh
authors.affiliation: Department of Medical Genetics, University of British Columbia, Vancouver, British Columbia, Canada; Child & Family Research Institute, Vancouver, British Columbia, Canada.
authors.forename: Maria S
authors.initials: MS
authors.lastname: Peñaherrera
authors.affiliation: Department of Medical Genetics, University of British Columbia, Vancouver, British Columbia, Canada; Child & Family Research Institute, Vancouver, British Columbia, Canada.
authors.forename: Wendy P
authors.initials: WP
authors.lastname: Robinson
mesh_terms: D000199:Actins / Q000235:genetics; D001324:Autoantigens / Q000235:genetics; D004247:DNA / Q000097:blood* / Q000235:genetics / Q000302:isolation & purification; D005260:Female; D005865:Gestational Age; D006801:Humans; D011247:Pregnancy; D011248:Pregnancy Complications / Q000097:blood* / Q000235:genetics; D060888:Real-Time Polymerase Chain Reaction; D043262:Ribonuclease P / Q000235:genetics; D014327:Trophoblasts / Q000378:metabolism; D025521:Tumor Suppressor Proteins / Q000235:genetics
chemical_list: D000199:Actins; D001324:Autoantigens; C411762:RASSF1 protein, human; C104646:RPP30 protein, human; D025521:Tumor Suppressor Proteins; D004247:DNA; D043262:Ribonuclease P