| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
|
|
|
|||||||
|
|||||||||
Letters to the Editor |
1 University Women’s Hospital, Department of Research, Basel, Switzerland
2 Center for Research in Biomedicine, UWE Bristol Genomics, Research Institute, University of the West of England, Bristol, United Kingdom
aAddress correspondence to this author at: University Women’s Hospital/Department of Research, Spitalstrasse 21, CH 4031 Basel, Switzerland. Fax 41-61-265-9399; e-mail shahn{at}uhbs.ch.
To the Editor:
Cell-free fetal DNA in the maternal circulation holds great potential for noninvasive prenatal diagnosis (1)(2). However, the manual isolation of cell-free DNA from plasma can be a time-consuming process, and current techniques involving spin columns usually require multiple reloadings of the columns with sample material, increasing the risk of contamination (3)(4)(5). Use of an automated system for DNA isolation can reduce the time involved, increase the throughput, and help reduce inconsistencies resulting from human error in processing of different plasma samples. We compared 2 techniques for extracting cell-free DNA from maternal plasma: the MagNA PureTM LC Instrument (Roche Applied Science), an automated DNA isolation system; and a frequently used manual technique involving spin-column technology (High Pure PCR Template Preparation KitTM; Roche Diagnostics).
After approval was granted from the Cantonal Institutional Review Board of Basel, we obtained 9 plasma samples from women carrying male fetuses (first to third trimester) to test the methods of DNA extraction. Identical samples were used for both methods, and the plasma was prepared according to our standard protocol and stored at –70 °C (4)(5). For the manual extraction method, cell-free DNA was extracted from 400 µL of plasma according to the manufacturer’s instructions and eluted with 100 µL of elution buffer. For the automated method, DNA was extracted from 1000 µL of plasma by the Roche MagNA Pure LC DNA Isolation Kit–Large Volume and eluted with 200 µL of elution buffer. On the following day, we processed a second identical set of samples, using the same procedures as described above, which gave a total of 18 samples tested per method.
Total cell-free DNA was quantified by a real-time PCR assay for the ubiquitous glyceraldehyde-3-phosphate dehydrogenase (GAPDH) gene, whereas the amount of cell-free fetal DNA was assessed by use of the multicopy DYS14 sequence (6). All concentrations are expressed as genome-equivalents (GE)/mL of maternal plasma (3).
Our analysis revealed that manual extraction yielded 23.4% more total cell-free DNA than the automated method [mean (range), 2890 (797–10 060) GE/mL vs 2215 (620–9010) GE/mL]. However, when we examined the quantity of cell-free fetal DNA obtained, we found that the automated system yielded 40.7% more cell-free fetal DNA than the manual method [mean (range), 86 (40–204) GE/mL vs 51 (9–160) GE/mL].
Another surprising finding was that the amplification efficiencies of the late cycles of the real-time PCR reactions appeared to be affected by the method used for DNA isolation, in that the steepness of the amplification curve was generally lower for the manually prepared samples than for the automated samples. This phenomenon became more evident when the amplification efficiencies of the late cycles were calculated from the slope of the amplification plots by the following equation:
 |
). The mean efficiency of the amplification reactions for the DYS14 locus for the automated preparations was 66%, whereas that for the manually prepared samples was 46% (Fig. 1B
). This finding may be attributable to the more thorough removal of inhibitors in the plasma samples by the automated DNA extraction method compared with the manual extraction method. The amplification efficiencies of diluted genomic DNA calibrators were 
90% for both assays.
|
In summary, our data from this small exploratory study indicate that the extraction of cell-free DNA by automated means is not only less labor-intensive and more amenable to high-throughput analysis but may indeed provide material that is optimal for PCR analysis, in that it may contain fewer inhibitors. The more efficacious retrieval of cell-free fetal DNA compared with maternal DNA may reflect the molecular size differences that exist between maternal and fetal cell-free DNA (4)(7). These aspects could be investigated further in a larger study.
References
The following articles in journals at HighWire Press have cited this article:
|
|
 |
|
  Y. Li, W. Holzgreve, V. Kiefer, and S. Hahn MALDI-TOF Mass Spectrometry Compared With Real-Time PCR for Detection of Fetal Cell-Free DNA in Maternal Plasma Clin. Chem., December 1, 2006; 52(12): 2311 - 2312. [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
