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Technical Briefs |
Divisions of1 Newborn Medicine and 2 Genetics, Department of Pediatrics, Floating Hospital for Children, and3 Institute of Clinical Research and Health Policy Studies, Tufts-New England Medical Center, Boston, MA;
aaddress correspondence to this author at: Tufts-New England Medical Center, 750 Washington St., Box 394, Boston MA 02111; fax 617-636-1469, e-mail dbianchi{at}tufts-nemc.org
Much current research focuses on the properties and clinical applications of circulating nucleic acids (1). The recent discovery of cell-free RNA in the plasma and serum of cancer patients has generated considerable interest (2)(3)(4)(5)(6). Circulating RNA is surprisingly stable, and Ng et al. (7) recently showed that a considerable proportion of plasma mRNA species is particle associated and thus possibly protected from nuclease degradation (8). Fetal-derived mRNA has also been found in the plasma and serum of pregnant women (9)(10)(11) and in amniotic fluid (12), and has many potential clinical applications (13)(14). Amniotic fluid is routinely collected during amniocentesis for fetal chromosome analysis or fetal lung maturity studies. However, little is known regarding the biology of circulating fetal mRNA or fetal mRNA in amniotic fluid.
In this report, we explore whether cell-free fetal nucleic acids in amniotic fluid have properties similar to circulating DNA and mRNA. Expanding on the work of Ng et al. (7), we hypothesized that cell-free fetal mRNA in amniotic fluid might be present in a particle-associated form and could thus be filterable, whereas the non-particle–associated form of DNA would be present in such high concentrations that there would be no significant reduction in its quantity by filtration. Additionally, we wished to compare the quantities of nucleic acids in amniotic fluid containing cells with the quantities in filtered and unfiltered cell-free supernatant. We hypothesized that whole amniotic fluid containing amniocytes would contain a significantly larger amount of DNA and RNA than cell-free amniotic fluid.
This study was performed with Institutional Review Board approval from Tufts-New England Medical Center. Seven amniotic fluid samples with a minimum volume of 3 mL each were obtained from women undergoing scheduled amniocenteses. One sample originated from a woman with polyhydramnios undergoing therapeutic amnioreduction. From 5 of the 7 samples, two 200-µL portions of uncentrifuged, unfiltered amniotic fluid were set aside at –80 °C. Uncentrifuged fluid was not available for the other 2 samples because the amniocytes were needed for clinical studies. For all 7 samples, the remaining amniotic fluid was aliquoted into 1.5-mL microcentrifuge tubes and centrifuged at 1600g for 10 min at 4 °C. The supernatant was then carefully removed and subjected to a second centrifugation at 16 000g for 10 min at 4 °C. The supernatant was again carefully removed and then divided into 4 additional aliquots: 3 portions were individually passed through filters (Millex-GV; Millipore) with pore sizes of 0.22, 0.45, and 5 µm. The remaining aliquot was not subjected to filtration. All aliquots were then divided into 2 portions and stored at –80 °C until RNA and DNA extractions were performed.
For each sample, RNA was extracted from 200 µL of each of the 5 amniotic fluid aliquots (uncentrifuged, cell-free unfiltered, and portions passed through filters with pore sizes of 0.22, 0.45, and 5 µm) with the QIAamp Viral RNA Mini Kit (Qiagen), according to the "Viral RNA Mini Spin Protocol" as recommended by the manufacturer. The buffer volumes were adjusted proportionally for sample size. A 15-min incubation at room temperature with RNase-free DNase (Qiagen) was used between buffers AW1 and AW2. RNA was stored at –80 °C until analysis.
For each sample, DNA was extracted from 200 µL of each of the 5 amniotic fluid aliquots described above with the QIAamp DNA Mini Kit (Qiagen), according to the "Blood and Body Fluid Spin Protocol" as recommended by the manufacturer. DNA was stored at –80 °C until analysis.
Real-time quantitative reverse transcription-PCR was used to measure the mRNA concentration in amniotic fluid, with transcript quantification verified by parallel amplification of the glyceraldehyde-3-phosphate dehydrogenase (GAPDH) gene, as described previously (7).
Real-time quantitative PCR was used to measure the DNA concentration in amniotic fluid, with transcript quantification verified by parallel amplification of the ß-globin gene, as described previously by Lo et al. (15) except that each primer was used at 100 nM and the probe was used at 50 nM.
Amplification data were collected by the 7700 Sequence Detector and analyzed with the Sequence Detection System software, Ver. 1.6.3 (PE-ABI). Each sample was run in triplicate with the mean results of the 3 reactions used for further calculations.
Descriptive statistics, including medians and 25th and 75th percentile ranges, were generated for GAPDH mRNA and ß-globin DNA in amniotic fluid separately in 5 aliquots: uncentrifuged, cell-free unfiltered, and portions passed through filters with pore sizes 0.22, 0.45, and 5 µm. A nonparametric Friedman 2-way ANOVA test was carried out to compare differences in GAPDH mRNA and ß-globin DNA concentrations between aliquots. As a follow-up procedure to compare effects of filter sizes in a pairwise manner with an adjustment for multiple comparisons, we used the Student–Newman–Keuls test with prior logarithmic transformation of the studied outcomes. The threshold for significance was set at 
= 0.05. All statistical tests were performed with SAS/STAT software (SAS Institute, Inc.).
The decrease in GAPDH mRNA concentration in amniotic fluid samples with cell removal and filtration with decreasing pore size is shown in Fig. 1A
(Friedman test, P = 0.0001). Pairwise analysis showed a statistically significant difference between the samples filtered with 0.22 µm pore size and the rest (Student–Newman–Keuls test, P <0.05). Overall, the GAPDH mRNA concentration decreased by a median of more than 60-fold (interquartile range, 27- to 100-fold) in comparisons of paired samples from the uncentrifuged samples and the portions passed through the 0.22 µm filters. The GAPDH mRNA concentration decreased by a median of more than 17-fold (interquartile range, 12- to 19-fold) in comparisons of paired samples from the centrifuged, unfiltered samples and the portions passed through the 0.22 µm filters. In comparison, there was no statistically significant difference in ß-globin DNA concentrations (Friedman test, P = 0.98) except for the uncentrifuged fraction vs the rest (Fig. 1B
). Pairwise analysis confirmed the statistically significant difference between the uncentrifuged fraction vs the rest (Student–Newman–Keuls test, P <0.05). The ß-globin DNA concentration decreased by a median of more than 32-fold (interquartile range, 10- to 50-fold) in comparisons of the paired samples from the uncentrifuged samples and the portions passed through the 0.22 µm filters.
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The study of cell-free RNA, particularly fetal RNA in the maternal circulation, and RNA in amniotic fluid is a new field of interest with many potential clinical applications (12)(13)(14). However, very little is known about the kinetics and origin of cell-free mRNA. Apoptosis has been suggested as a source of these nucleic acids (16) and could explain the remarkable stability of cell-free mRNA as a result of packaging into protected apoptotic bodies (17). Ng et al.(7) recently explored the properties of nucleic acids in plasma and showed that filterable GAPDH mRNA species are present, and therefore likely to be particle bound, whereas the majority of ß-globin DNA is not filterable and thus is not particle bound.
Our study analyzed cell-free nucleic acids in amniotic fluid for the presence of particle-associated mRNA species, and like Ng et al. (7), we found the greatest decrease in GAPDH mRNA after filtration through a 0.22 µm filter, whereas filtration did not significantly reduce the amount of cell-free ß-globin DNA present. Additionally, this study evaluated the difference in amounts of nucleic acids present in whole amniotic fluid containing cells and the cell-free fraction. Much more ß-globin DNA could be isolated from whole amniotic fluid than from the cell-free fraction, but there was no statistically significant difference in the amount of GAPDH mRNA that could be isolated from the two fractions.
This study demonstrates similar properties of cell-free nucleic acids in amniotic fluid and in plasma. Ng et al. (7) suggested that circulating DNA and RNA in plasma are both protected from degradation by associated particles, but the non-particle–associated form of DNA is less efficiently degraded than the non-particle–associated form of RNA and thus is present in much greater quantities relative to the particle-associated form. We extend this hypothesis to the properties of cell-free nucleic acids in amniotic fluid. These data suggest that in different body fluids, there is a universal mechanism of cell-free nucleic acid processing, possibly via packaging during apoptosis.
To our knowledge, this is the first study to evaluate the properties of cell-free mRNA in amniotic fluid. We have also studied fetal gene expression in amniotic fluid (12). More research is needed to further evaluate the origin and kinetics of cell-free nucleic acids in amniotic fluid as this material has significant clinical potential for the study of health and development in living fetuses.
References
The following articles in journals at HighWire Press have cited this article:
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  D. K. Slonim, K. Koide, K. L. Johnson, U. Tantravahi, J. M. Cowan, Z. Jarrah, and D. W. Bianchi Functional genomic analysis of amniotic fluid cell-free mRNA suggests that oxidative stress is significant in Down syndrome fetuses PNAS, June 9, 2009; 106(23): 9425 - 9429. [Abstract] [Full Text] [PDF]
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 O. Lapaire, D. W. Bianchi, I. Peter, B. O'Brien, H. Stroh, J. M. Cowan, U. Tantravahi, and K. L. Johnson Cell-Free Fetal DNA in Amniotic Fluid: Unique Fragmentation Signatures in Euploid and Aneuploid Fetuses Clin. Chem., March 1, 2007; 53(3): 405 - 411. [Abstract] [Full Text] [PDF]
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indicate data points outside the 10th and 90th percentiles. The * in panel B denotes a single outlier at 
116 000 genome-equivalents/mL in the unspun data set; this outlier was removed to allow for clarity of data presentation.