OBJECTIVES: To investigate the performance of an automated system for the extraction of cell-free DNA of maternal and fetal origin from stored serum samples for subsequent quantitative real-time polymerase chain reaction (PCR) analysis. METHODS: Thirty-two maternal blood samples between the early second trimester and term were obtained. Cell-free DNA was extracted from replicate stored sera using a column-based manual isolation procedure and with an automated system, the MagNA Pure LC Instrument. Real-time quantitative PCR for the ubiquitous glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and male-specific DYS14 loci was performed. RESULTS: The extraction yields for both total and fetal DNA and the quality of the purified nucleic acids were similar for the automated system or the manual procedure. However, the number of false-negative results in samples collected early in pregnancy was reduced with the automated extraction. Furthermore, the extraction rate by the automated system was highly reproducible over time. CONCLUSIONS: We validated the use of an automated extraction system for the isolation of fetal DNA from stored serum. This procedure might be exploited in the future for high-throughput non-invasive fetal gene analysis of archived serum samples.
OBJECTIVES: To investigate the performance of an automated system for the extraction of cell-free DNA of maternal and fetal origin from stored serum samples for subsequent quantitative real-time polymerase chain reaction (PCR) analysis. METHODS: Thirty-two maternal blood samples between the early second trimester and term were obtained. Cell-free DNA was extracted from replicate stored sera using a column-based manual isolation procedure and with an automated system, the MagNA Pure LC Instrument. Real-time quantitative PCR for the ubiquitous glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and male-specific DYS14 loci was performed. RESULTS: The extraction yields for both total and fetal DNA and the quality of the purified nucleic acids were similar for the automated system or the manual procedure. However, the number of false-negative results in samples collected early in pregnancy was reduced with the automated extraction. Furthermore, the extraction rate by the automated system was highly reproducible over time. CONCLUSIONS: We validated the use of an automated extraction system for the isolation of fetal DNA from stored serum. This procedure might be exploited in the future for high-throughput non-invasive fetal gene analysis of archived serum samples.
Authors: D A Khodakov; D D Mamaev; E I Dementieva; I V Filatov; D A Yurasov; A I Cherepanov; V A Vasiliskov; O V Smoldovskaya; D A Gryadunov; V M Mikhailovich; A S Zasedatelev; A A Makarov Journal: Dokl Biochem Biophys Date: 2010-12-24 Impact factor: 0.788