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Letters to the Editor |
1 Department of Chemical Pathology, The Chinese University of Hong Kong, Hong Kong SAR
2 University Women’s Hospital, Department of Research, Basel, Switzerland
aAddress correspondence to this author at: Department of Chemical Pathology, The Chinese University of Hong Kong, Room 38023, 1/F Clinical Sciences Building, Prince of Wales Hospital, 30-32 Ngan Shing St., Shatin, New Territories, Hong Kong Special Administrative Region, China. Fax 852-2194-6171; e-mail loym{at}cuhk.edu.hk.
To the Editor:
Detection of fetal-derived DNA and RNA molecules in maternal plasma is a promising approach for noninvasive prenatal diagnosis (1). Analysis of circulating fetal RNA, unlike analysis of fetal Y-chromosomal DNA, can be used in pregnancies with fetuses of either gender(2). Placenta-derived mRNA species are readily detectable in maternal plasma(2). The fetal hematopoietic compartment may be another source of nucleic acids in maternal plasma(3). Wataganara et al.(4) reported detection of 
-globin mRNA in maternal plasma. However, expression of -globin is not a fetus-specific phenomenon and is shared by maternal erythroid cells(5), and -globin mRNA is readily detectable in nonpregnant individuals(4). In this study, we assessed whether rhesus D (RHD gene) mRNA derived from fetal erythroid cells is detectable in the plasma of rhesus D-negative pregnant women.
Fifteen rhesus D-negative women at 11–40 weeks of gestation (Table 1
), attending the University Women’s Hospital, Basel, were recruited with informed consent and institutional ethics approval. Trisomy 21 was subsequently confirmed in 1 pregnancy, which was excluded from the analysis. We collected 15 mL of maternal peripheral blood into EDTA tubes. Cord blood was collected from 3 of the term pregnancies after delivery. The blood samples were stored at 4 °C until processing within 3 h by centrifugation (2). Trizol LS reagent (4 mL) was mixed with 3.2 mL of plasma and stored frozen at –80 °C. The plasma samples were sent to Hong Kong in dry ice.
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The maternal plasma and cord blood samples were analyzed for RHD, glyceraldehyde-3-phosphate dehydrogenase (GAPDH), and human placental lactogen (hPL) mRNA by 1-step real-time quantitative reverse transcription-PCR assays. The latter 2 assays had been described previously (2). The RHD mRNA assay was designed by use of Primer Express software, Ver. 2.0 (Applied Biosystems), with the fluorescent probe crossing the junction between exons 7 and 8 (GenBank accession no. BN000065). Specificity of the assay for RHD was conferred by the forward primer. The primer and probe sequences were as follows: forward primer, 5'-TGC TTG ATA CCG TCG GAG C-3'; reverse primer, 5'-TGA GTT CCC CAA TGC TGA GG-3'; fluorescent probe, 5'-(FAM) AAT GGC ATG ATT GGC TTC CAG GTC C (TAMRA)-3', where FAM is 6-carboxyfluorescein and TAMRA is 6-carboxytetramethylrhodamine. The reactions were set up according to the manufacturer’s instructions (EZ rTth RNA PCR reagent set; Applied Biosystems) in a reaction volume of 25 µL with 200 nM of each primer and 50 nM of the fluorescent probe. We used 5 µL of extracted plasma RNA for each reaction, which was performed in a thermal cycler with fluorescent detector (ABI Prism 7900; Applied Biosystems) at 50 °C for 2 min for the activation of uracil-N-glycosylase, 60 °C for 30 min for reverse transcription, and 95 °C for 5 min, followed by 45 cycles of 94 °C for 20 s and 1 min at 60 °C. The calibration curve was constructed with serial dilutions of an HPLC-purified single-stranded synthetic DNA oligonucleotide (5'-CTG GTG CTT GAT ACC GTC GGA GCC GGC AAT GGC ATG ATT GGCTTC CAG GTC CTC CTC AGC ATT GGG GAA CTC AGC TT-3') at 2.5 x 107 to 2.5 copies. Specificity of the assay was confirmed by the lack of amplification for buffy coat RNA of rhesus D-negative individuals. Sensitivity of the assay was assessed by use of buffy coat and plasma RNA from 10 rhesus D-positive nonpregnant individuals. Positive amplification was noted from all samples, and the assay could detect 5 copies/mL of plasma.
Sample analysis was performed without knowledge of the fetal rhesus D status. GAPDH mRNA was detectable in all samples, whereas hPL mRNA, a placenta-specific transcript (2), was detectable in all maternal plasma samples (data not shown). RHD mRNA was detected in the cord plasma sample collected from a rhesus D-positive neonate, but in no maternal plasma samples (Table 1
).
Although RHD mRNA has been detected in fetal erythroid cells isolated from maternal circulation (6)(7), we found no RHD mRNA in maternal plasma. We suggest that fetal erythroid cells are unlikely to contribute a large portion of the fetal mRNA in maternal plasma. We believe that detection of plasma RHD mRNA in rhesus D-negative pregnant women is a more specific approach than is the study of -globin, which is expressed by both pregnant and nonpregnant individuals(4) and is highly expressed in placental tissues (accession no. GSM18967; GEO DataSets, www.ncbi.nlm.nih.gov). This latter phenomenon may explain the increased concentrations of -globin mRNA found in maternal plasma and the significant reductions after pregnancy termination(4). In summary, our data do not suggest that fetal erythroid cells contribute a predominant fraction of fetal mRNA in maternal plasma.
Acknowledgments
This work is supported by an Earmarked Research Grant from the Hong Kong Research Grants Council (CUHK 4277/04M) and by the Direct Grant Scheme of the Chinese University of Hong Kong.
References
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