Time Profile of Appearance and Disappearance of Circulating Placenta-Derived mRNA in Maternal Plasma

Abstract

The ranges of the RQ values were calculated by use of the equation: RQ ϭ 2 Ϫ⌬⌬Ct , with ⌬⌬Ct ϩ SD and ⌬⌬Ct Ϫ SDs. The Micro Fluidic Cards detect a 2-fold difference in gene expression at the 99.7% confidence level. The efficiencies of all target and reference amplifications were nearly identical as analyzed by serial dilutions using 2, 1, 0.5, 0.25, and 0.125 ng of calibrator cDNA. When we plotted the log input amount of cDNA vs the ⌬Ct values, the slope was Ͻ0.1. The correlation coefficients were always Ͼ0.95, which is comparable to our previously established method (5 ). We assessed the within-run and day-to-day imprecision of our assay by measuring 4 identical samples in duplicate; the CVs were Ͻ1.6% and Ͻ2.9%, respectively.As can be seen in Table 1, 30 ABC transporters displayed detectable mRNA expression in human primary monocytes, based on ⌬Ct values Ͼ25. Twenty-six genes were induced more than 2-fold during macrophage differentiation, and 12 genes were sensitive to LXR/RXR agonist incubation, with 10 up-regulated ABC transporters and only 2 down-regulated genes. Although the differentiation-dependent and sterol-regulated induction of ABCA1 and ABCG1 is well established (7 ), parallel transcript profiling, using our Human ABC Transporter TaqMan Low-Density Array, revealed several additional differentiation-dependent ABC transporters and novel LXR/RXR-regulated ABC transporters, including ABCB1 (MDR1), ABCB9, ABCB11 (BSEP), ABCC2 (MRP2), ABCC5 (MRP5), ABCD1 (ALD), ABCD4, and ABCG2. Despite the large dynamic range of gene regulation (e.g., a 24.5-fold induction of ABCA1 by 9-cis RA/T0901317), the range of differential transcript expression between 3 different donors was quite narrow (21.9 -27.4). This implies that the interindividual differences in response are much smaller than the differentiation-dependent and lipid-regulated effects, allowing screening processes with a limited number of probands.These findings are particularly relevant for lipidomics and cardiovascular research, as these ABC transporters are novel candidates for lipid disorders and pharmacologic targets for lipid-modulating drugs.In summary, we have developed a Human ABC Transporter TaqMan Low-Density Array based on the TaqMan chemistry, the Micro Fluidic Card, and the 7900HT Sequence Detection System. Compared with standard TaqMan reverse transcription-PCR methods, the Micro Fluidic Card requires less sample material (2 ng instead of 50 ng of total RNA-equivalents per gene), only one-tenth the volume of TaqMan Universal Master Mix, and much less hands-on time. This assay could be a useful tool for monitoring dysregulated ABC transporter mRNA profiles in human lipid disorders and cancer-related multidrug resistance. Furthermore, the pharmacologic and metabolic regulation of ABC transporter gene expression important for drug development could be analyzed in large screening approaches using this Human ABC Transporter TaqMan Low-Density Array.We thank Ernst Arnoldi, Astrid Potratz, and Andrea Geiger for support and discussions, and Manfred H...

Integrative single-cell and cell-free plasma RNA transcriptomics elucidates placental cellular dynamics

“…Previous maternal plasma transcriptomic profiling studies have shown that certain trophoblast-specific transcripts and the overall fractional placental contribution increase with gestation (20,39,40). The fraction of fetal-derived RNA increases from only 3.7% in early pregnancy to 11.28% in late pregnancy (19,20).…”

Gene expression analysis in pregnant women and their infants identifies unique fetal biomarkers that circulate in maternal blood

“…Since fetal-derived mRNA is rapidly cleared from maternal circulation following delivery (10,11), antepartum and postpartum samples were compared with paired newborn umbilical cord blood samples to identify unique fetal biomarkers in maternal whole blood. Our hypothesis was that if specific gene transcripts were detected in pregnant women prior to delivery as well as in their infants' cord blood, yet were significantly reduced or absent in maternal blood within 24 to 36 hours of delivery, these gene transcripts could theoretically originate from the fetus (Figure 1).…”

mRNA of Placental Origin in Maternal Serum of Women with Normal and Preeclamptic Pregnancies

“…Placental RNA was detected as early as the 4th week of gestation and rapidly cleared postpartum [13] , and placenta-specific RNA transcripts coding for hPL, ␤ -hCG and CRH have been detected in maternal blood of normal pregnant women and in those affected by trophoblast diseases [14] . Finally, Lo et al [32] analyzed the allelic ratio of a single nucleotide polymorphism of the placenta-derived placenta-specific 4 (PLAC4) mRNA in maternal plasma and Concu et al [33] demonstrated that mRNA provides a gender-independent marker to distinguish between normal and pathological pregnancies by measuring rapid clearance of mRNA for PLAC1 gene in maternal blood after delivery .…”

“…Besides recent findings in the study of circulating DNA, there is growing interest in the diagnostic potential of circulating RNA. Placental RNA can be detected as early as the 4th week of gestation and is rapidly cleared postpartum [13] . As already shown for DNA, the placenta has been suggested to be the main source of circulating RNA.…”