Fetal rhesus D mRNA is not detectable in maternal plasma.

Detection of fetal-derived DNA and RNA molecules in maternal plasma is a promising approach for noninva-sive 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 non-pregnant individuals (4). In this study, we assessed whether rhesus D (RHD gene) mRNA derived from fe-tal 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 Ϫ80 °C. The plasma samples were sent to Hong Kong in dry ice. 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: for