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Clinical Chemistry 51: 1786-1795, 2005. First published August 4, 2005; 10.1373/clinchem.2005.052340
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(Clinical Chemistry. 2005;51:1786-1795.)
© 2005 American Association for Clinical Chemistry, Inc.

Molecular Diagnostics and Genetics

Circulating Placental RNA in Maternal Plasma Is Associated with a Preponderance of 5' mRNA Fragments: Implications for Noninvasive Prenatal Diagnosis and Monitoring

Blenda C.K. Wong1, Rossa W.K. Chiu1, Nancy B.Y. Tsui1, K.C. Allen Chan1, Lin W. Chan2, Tze K. Lau2, Tse N. Leung2 and Y.M. Dennis Lo1,a

Departments of1 Chemical Pathology and 2 Obstetrics and Gynecology, The Chinese University of Hong Kong, Prince of Wales Hospital, Shatin, New Territories, Hong Kong SAR, China.

aAddress correspondence to this author at: Department of Chemical Pathology, The Chinese University of Hong Kong, Prince of Wales Hospital, Rm 38023, 1/F Clinical Sciences Bldg, 30-32 Ngan Shing Street, Shatin, New Territories, Hong Kong SAR, China. Fax 852-2194-6171; e-mail loym{at}cuhk.edu.hk.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Background: The molecular characteristics of placental RNA circulating in maternal plasma are unknown. We investigated the integrity of circulating placental RNA in maternal plasma and tested the relevance of plasma RNA integrity for noninvasive prenatal diagnosis.

Methods: Six different placental transcripts and mRNA of the housekeeping gene glyceraldehyde-3-phosphate dehydrogenase (GAPDH) were quantified for the 5' and 3' regions in maternal plasma by 1-step real-time reverse transcription-PCR (RT-PCR) assays. This quantitative strategy was validated by 2-step RT-PCR and serial dilution experiments. The rates of detection by the 5' and 3' assays for the ß-subunit of human chorionic gonadotropin (ßhCG) were assessed in maternal plasma samples collected from different gestational periods.

Results: For 5 of the 7 genes, the plasma mRNA concentrations measured by the 5' amplicons were significantly higher than those measured by the corresponding 3' amplicons. Every transcript under study demonstrated a higher rate of detection in the 5' assay than in the 3' assay in maternal plasma. In particular, the detection rate of ßhCG mRNA in maternal plasma was increased throughout gestation when the 5' assay was used.

Conclusions: Circulating placental RNA is associated with a preponderance of 5' mRNA fragments in maternal plasma. Apart from its intrinsic biological interest, this information could have important implications for the development of new assays targeting fetal RNA markers for noninvasive prenatal diagnosis and monitoring.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Cell-free circulating RNA has recently opened up new possibilities of noninvasive gene expression profiling (1). The discovery of tumor-derived RNA in the plasma/serum of cancer patients has revealed clinical potential for the early detection and monitoring of malignant diseases (2)(3)(4)(5)(6). Beyond the field of oncology, demonstration of the presence of fetal RNA in the plasma of pregnant women (7) has provided a feasible alternative for noninvasive prenatal investigation. In this area, our group has recently shown that the placenta is an important source of fetal RNA in maternal plasma, which offers the potential advantage of being usable irrespective of the gender or genetic polymorphism differences between the pregnant woman and her fetus when compared with fetal DNA analysis (8). Further study has led to the development of a systematic and high-throughput strategy based on microarray analysis to identify new placental RNA markers for detection in maternal plasma (9). Of clinical significance, quantitative aberrations of placental transcripts in maternal circulation have been shown to be associated with preeclampsia (10) and fetal chromosomal aneuploidies (11). These findings provide direct evidence that the measurement of fetal RNA in maternal plasma may represent a new approach for assessing fetal gene expression under different pathologic or physiologic conditions.

Despite the rapid development of cell-free circulating RNA as a diagnostic tool, much of the biology of these molecular species remains to be elucidated. In view of the well-known lability of RNA and the existence of ribonucleases in the circulation (12)(13), the recent demonstration of stable RNA in plasma and serum (14) came as a surprising and intriguing finding for the field of medical diagnostics. It has been proposed that the stability of such extracellular RNA is conferred by its association with certain types of particulate matter, e.g., apoptotic bodies (15)(16). Subsequent studies have also unveiled the molecular aspects of plasma nucleic acids. It was shown that Epstein–Barr virus DNA molecules in the plasma of cancer patients consisted mainly of short DNA fragments <180 bp in length (17) and that fetal DNA species in the plasma of pregnant women were relatively shorter than the maternal counterparts (18)(19). However, no information is currently available regarding the integrity of plasma RNA.

In this study, we investigated the integrity of plasma RNA by developing real-time quantitative reverse-transcription-PCR (RT-PCR) 1 assays to target multiple regions along a transcript. Whether a transcript is intact or not would depend on the observed abundances of the different amplicons from the corresponding transcript. In particular, we focused on measurement of the 5', middle, and 3' regions of a transcript. A discrepancy in the measured quantities or detection rates of the 3 regions would suggest the presence of incomplete mRNA fragments. We first analyzed the 5', middle, and 3' regions of the glyceraldehyde-3-phosphate dehydrogenase (GAPDH) transcript in the plasma of healthy persons. This quantitative strategy was validated by 2-step RT-PCR and serial dilution experiments. We then applied the same approach and used the placenta as a model system for studying the integrity of circulating placental RNA. As a panel, GAPDH mRNA and 6 different placental transcripts were systemically quantified for the 5', middle, and 3' regions in placental tissues and for the 5' and 3' regions in the plasma of pregnant women. These transcripts previously were shown to have differential expression in the placenta by microarray analysis (9), including the ß-subunit of human chorionic gonadotropin (ßhCG), placenta-specific 1 (PLAC1), tissue factor pathway inhibitor 2 (TFPI2), adrenomedullin (ADM), inhibin-ß A subunit (INHBA), and pregnancy-associated plasma protein A (PAPPA). Lastly, to demonstrate the clinical relevance of plasma RNA integrity for noninvasive prenatal diagnosis, we compared the rates of detection for the 5' and 3' ßhCG assays, using maternal plasma samples from different gestational ages.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
participants
Peripheral blood samples were collected from 10 healthy nonpregnant persons and 74 healthy pregnant women. The latter group consisted of 8 first-trimester, 25 second-trimester, and 41 third-trimester pregnancies. Term placental samples were obtained from another 10 healthy pregnant women undergoing caesarian sections. All pregnant women were recruited with informed consent at the Department of Obstetrics and Gynecology, Prince of Wales Hospital, Hong Kong. The study was approved by the Clinical Research Ethics Committee of The Chinese University of Hong Kong.

processing of blood and tissue samples
Blood samples were collected into EDTA-containing tubes and centrifuged at 1600g for 10 min at 4 °C (Centrifuge 5810R; Eppendorf). Plasma was then carefully transferred to plain polypropylene tubes to be recentrifuged at 16 000g for 10 min at 4 °C (Centrifuge 5415D; Eppendorf) for complete removal of residual cells (20). The supernatants were collected into fresh polypropylene tubes and mixed with TRIzol LS reagent (Invitrogen). Buffy coat was isolated after the first centrifugation step and mixed directly with the TRIzol LS reagent (Invitrogen). Placental samples were stored in an RNAlater (Ambion) stabilizing solution immediately after collection. The solution was removed after storage at 4 °C overnight. All samples were kept at –80 °C until RNA extraction.

rna extraction from plasma and buffy coat samples
For plasma RNA extraction, 1.6 mL of plasma was mixed with 2 mL of TRIzol LS reagent (Invitrogen) and 0.4 mL of chloroform (15). For buffy coat RNA extraction, 0.3 mL of buffy coat was mixed with 0.9 mL of TRIzol LS (Invitrogen) and 0.24 mL of chloroform, as suggested by the manufacturer. The mixture was separated into different phases by centrifugation at 12 000g for 15 min at 4 °C (Centrifuge 5417R; Eppendorf). The aqueous layer was then carefully transferred to fresh polypropylene tubes. For RNA precipitation, one volume of 700 mL/L ethanol was added to one volume of the aqueous layer. The mixture was applied to an RNeasy minicolumn (Qiagen) and processed according to the manufacturer’s protocols. Total RNA was eluted in 30 µL of RNase-free water and stored at –80 °C until use. DNase treatment was performed with either the RNase-Free DNase Set (Qiagen) or Amplification Grade DNase I (Invitrogen).

rna extraction from tissue samples
For tissue RNA extraction, the samples were homogenized in TRIzol reagent (Invitrogen) and mixed with chloroform according to the manufacturer’s instructions. Briefly, RNA was precipitated from the aqueous layer by mixing with isopropyl alcohol and washed with 750 mL/L ethanol. The samples were then purified with an RNeasy Mini Kit (Qiagen) according to the manufacturer’s protocols. Total RNA was eluted in 30 µL of RNase-free water and treated with Amplification Grade DNase I (Invitrogen).

generation of calibration curves
The use of a single "full-length" cDNA calibrator was important for quantification of different regions within a transcript because potential bias may otherwise result from the use of multiple calibration curves. In this study, DNA calibrators were synthesized by TA cloning. The GAPDH gene was amplified from human pooled organ cDNA (Clontech), whereas amplification of PLAC1, TFPI2, ADM, and INHBA was carried out with human placental cDNA (Clontech). To generate a full-length cDNA, we performed PCR with the forward primer of the 5' assay and the reverse primer of the 3' assay, using the Advantage cDNA Polymerase Mix (Clontech). Similarly, the ßhCG gene was reverse-transcribed from chorionic villus RNA by use of the SuperScript One-Step RT-PCR with Platinum Taq System (Invitrogen). PCR-amplified cDNA constructs were cloned into the pGEM T-Easy Vector according to the manufacturer’s instructions (Promega). Plasmid DNA was isolated with a Wizard Plus Minipreps DNA Purification Kit (Promega) and confirmed by direct sequencing (BigDye Terminator, Ver. 3.1; Applied Biosystems). Calibration curves were constructed by use of serial dilutions of the extracted plasmid DNA.

Cloning of a full-length cDNA was difficult for the PAPPA gene, which encodes a relatively long mRNA sequence (11 kb). As an alternative method, the targeted regions of the PAPPA transcript were prepared separately by PCR using human placental cDNA (Clontech). The primer sequences for the middle portion were 5'TTCAAGACTCAGTGTACCCAGGAT-3' (sense) and 5'-ACGCACGGTCACGTTCATT-3' (antisense). The primer sequences for the 5' portion were 5'-TAGAAGCTT-GGTCTCCGGCAGTGATCA-3' (sense), which introduced a HindIII restriction site, and 5'-TAGACTAGTCACCATTCACATAGAGCTTCATGA-3' (antisense), which introduced a SpeI restriction site. The primer sequences for the 3' portion were 5'-TAGCTCGAGTTCTCTTGGTCTTATTCCCATCCT-3' (sense), which introduced an XhoI restriction site, and 5'-TAGTCTAGACAGACAGTGTTATCAAATCCCACTC-3' (antisense), which introduced an XbaI restriction site. The middle construct was first inserted into the TOPO TA cloning vector (Invitrogen) at the TA cloning site. The 5'- and 3'-specific PCR constructs were digested at both ends with the appropriate restriction enzymes (BioLabs) and cloned between the HindIII and SpeI sites and between XhoI and XbaI sites of the vector, respectively. The plasmid product was extracted and confirmed by direct sequencing, as described above. The resulting plasmid vector contained all 3 targeted regions of the PAPPA transcript, suitable to be used as quantitative calibrators for each corresponding assay.

real-time quantitative rt-pcr
We used 1-step real-time quantitative RT-PCR to measure the mRNA concentrations of the following genes: GAPDH, ßhCG, PLAC1, TFPI2, ADM, INHBA, and PAPPA. Quantitative RT-PCR assays were designed to target the 5', middle, and 3' regions of each transcript. The sequence information of the primers and probes (Proligo; Helios) is given in Table S1 of the Data Supplement that accompanies the online version of this article athttp://www.clinchem.org/content/vol51/issue10/. The primer and probe sequences for the 5' regions of GAPDH and PLAC1 and for the middle region of ßhCG have been described previously (8)(9)(15). It should be noted that primer design across intron–exon junctions was not attainable for certain assays in this study, taking into consideration that each amplicon must target a particular region of a transcript (e.g., 5' and 3' ends). Nonetheless, precaution was taken to avoid the problem of genomic DNA contamination and to ensure the specificity of our RT-PCR assays. The tested RNA samples were subjected to DNase I treatment, as mentioned above, and controls were included to demonstrate the absence of DNA with the use of AmpliTaq Gold enzyme (Applied Biosystems) and omission of the reverse transcription step.

For valid data comparison in the analysis of plasma RNA, the different systems developed for each transcript were optimized with the corresponding DNA calibrator to acquire similar assay sensitivities. The lower limit of detection was 12 copies/mL for ADM; 24 copies/mL for TFPI2, INHBA, and PAPPA; and 48 copies/mL for GAPDH, ßhCG, and PLAC1.

The quantitative RT-PCR was set up according to the manufacturer’s instructions (EZ rTth RNA PCR Reagent Kit; Applied Biosystems) with a reaction volume of 50 µL. For the 5' assays, the primers were used at concentrations of 200 nM for GAPDH, ßhCG, and PLAC1; 300 nM for ADM and PAPPA; and 600 nM for TFPI2 and INHBA; the probes were used at concentrations of 80 nM for PLAC1; 100 nM for GAPDH, ßhCG, ADM, and PAPPA; and 200 nM for TFPI2 and INHBA. For amplification of the middle region, the primers were used at a concentration of 200 nM for all genes; the probes were used at concentrations of 80 nM for ßhCG and TFPI2 and 100 nM for the other genes. For the 3' assays, the primers were used at concentrations of 200 nM for GAPDH, ßhCG, TFPI2, and INHBA and 300 nM for PLAC1, ADM, and PAPPA. The probes were used at concentrations of 80 nM for TFPI2 and INHBA, and 100 nM for the other genes. For 5' and 3' amplification, extracted plasma RNA was used in volumes of 3 µL for GAPDH; 5 µL for ßhCG; 6 µL for ADM, 10 µL for TFPI2, PLAC1 and PAPPA; and 12 µL for INHBA. In addition, 3 µL of extracted plasma RNA was used for measuring the middle region of the GAPDH mRNA sequence. For each transcript, 0.5 ng of extracted placental RNA was used for quantification of the 3 targeted regions. For GAPDH and ßhCG, amplification was monitored and analyzed on an ABI Prism 7700 Sequence Detector (Applied Biosystems), whereas all other genes were studied on an ABI Prism 7900 Sequence Detector (Applied Biosystems). Multiple water blanks were included in every analysis. Each reaction mixture was incubated at 50 °C for 2 min to activate uracil N-glycosylase, followed by reverse transcription at 60 °C for 30 min. After an initial denaturation at 95 °C for 5 min, the mixture was cycled 45 times with denaturation at 94 °C for 20 s and 1 min of annealing and extension at 60 °C for GAPDH and ßhCG and 56 °C for TFPI2, PLAC1, ADM, INHBA, and PAPPA.

2-step rt-pcr
Peripheral blood samples from 10 healthy nonpregnant individuals were used in this experiment. Buffy coat RNA (20 ng) was reverse-transcribed with random and oligo(dT) primers (TaqMan Reverse Transcription Reagents; Applied Biosystems). For random priming, the cycling conditions were 25 cycles of 25 °C for 10 min, 48 °C for 30 min, and 95 °C for 5 min. For oligo(dT) priming, the cycling conditions were 25 cycles of 40 °C for 10 min, 48 °C for 30 min, and 95 °C for 5 min. The cDNA products were amplified by real-time quantitative PCR according to the manufacturer’s instructions (TaqMan PCR Core Reagent Kit; Applied Biosystems) for the 5' and 3' regions of the GAPDH transcript. The primers and probes were used at concentrations of 200 nM and 100 nM, respectively, and the calibrator was generated by TA cloning as described above. For amplification, 3 µL of cDNA was used in a reaction volume of 50 µL. The PCR conditions were 50 °C for 2 min for the activation of uracil N-glycosylase, followed by an initial denaturation at 95 °C for 10 min and then by 45 cycles of denaturation at 95 °C for 15 s and 1 min of annealing and extension at 60 °C.

serial dilution experiment
Buffy coat RNA samples, from 2 healthy nonpregnant individuals, with a starting concentration of 10 ng/µL were serially diluted 14 times with folds of dilution in the following order: 10-fold, 5-fold, and then 2-fold for 12 more dilutions. These diluted samples were assayed for the 5' and 3' GAPDH mRNA concentrations by 1-step real-time quantitative RT-PCR as described above

statistical analysis
Statistical analyses were performed using the SigmaStat 2.03 software (SPSS).


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
quantitative rt-pcr analysis of multiple regions of GAPDH MRNA in the plasma of healthy individuals
To investigate the integrity of plasma RNA, we measured the concentrations of the 5', middle, and 3' regions of the GAPDH transcript in the plasma of 10 healthy individuals, using 1-step real-time quantitative RT-PCR. The results are expressed as fractional mRNA concentrations relative to the absolute concentrations of the 5' amplicon to assess the degree of RNA integrity in plasma. As shown in Fig. 1A , there was a significant gradual decrease in the fractional GAPDH mRNA concentration (Friedman test, P <0.001) as the target site was shifted from the 5' to the 3' end of the transcript, inclusive of the middle region. Pairwise analysis further demonstrated a statistically significant difference for every pair of targeted regions (Student–Newman–Keuls test, P <0.05). The mRNA concentration of the 5' amplicon was 1.9-fold higher than that of the middle amplicon (middle-to-5' ratio = 0.53) and 20-fold higher than that of the 3' amplicon (3'-to-5' ratio = 0.051). These results suggested that there was a preponderance of 5' GAPDH mRNA fragments in the plasma of healthy individuals. Moreover, the 3 sets of quantitative data in terms of absolute mRNA concentrations were significantly correlated with one another [Pearson correlation analysis, r = 0.791 (P <0.01) for 5' end vs middle part; r = 0.689 (P <0.05) for 5' vs 3' ends; and r = 0.770 (P <0.01) for the middle part vs the 3' end; Fig. 1 , B–D], as expected for amplicons derived from the same transcript.



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  		Figure 1. Quantitative analysis of multiple regions of GAPDH mRNA in the plasma of healthy individuals as determined by 1-step real-time quantitative RT-PCR.

(A), fractional mRNA concentrations with respect to the absolute concentrations of the 5' region are plotted against amplicon location. The lines inside the boxes denote the medians. The boxes represent the interval between the 25th and 75th percentiles, and the whiskers represent the interval between the 10th and 90th percentiles. • denote outliers. (B), correlation between the absolute mRNA concentrations of the 5' and middle amplicons. (C), correlation between the absolute mRNA concentrations of the 5' and 3' amplicons. (D), correlation between the absolute mRNA concentrations of the middle and 3' amplicons. Given that all 3 assays had similar detection limits (48 copies/mL), cross-system comparison was valid in this experiment.

validation of 1-step real-time rt-pcr analysis of rna integrity in circulation
To exclude the possibility of technical bias in our 5' GAPDH RT-PCR system, we performed 2-step RT-PCR analysis of both the 5' and 3' GAPDH regions, using random and oligo(dT) primers for reverse transcription followed by gene-specific real-time PCR. The rationale for this experiment was based on the fact that the oligo(dT) primer could potentially introduce 3' end bias in the amplified products, as 3'-degraded transcripts would not be successfully reverse-transcribed. Demonstration of 3' mRNA predominance in the oligo(dT) primed system would suggest that our results were not attributable to any analytical bias favoring the 5' system. Buffy coat RNA samples from 10 healthy individuals were assayed for the 5' and 3' GAPDH mRNA concentrations by 2-step RT-PCR. As shown in Fig. 2A , the reaction using random primer produced a significantly higher concentration of 5' GAPDH amplicon (Wilcoxon test, P <0.01), consistent with our findings based on 1-step RT-PCR. In contrast, the reaction using oligo(dT) primer produced a significantly higher concentration of 3' GAPDH amplicon (Wilcoxon test, P <0.01), presumably as a result of 3' end bias during the reverse transcription step.



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  		Figure 2. Validation study of the overrepresentation of 5' GAPDH mRNA molecules, compared with 3' GAPDH molecules, in the circulation of healthy individuals.

(A), buffy coat concentrations of 5' and 3' GAPDH amplicons as determined by 2-step RT-PCR using random and oligo(dT) primers for reverse transcription. The lines inside the boxes denote the medians. The boxes represent the interval between the 25th and 75th percentiles, and the whiskers represent the interval between the 10th and 90th percentiles. • indicate outliers. (B and C), detection of 5' and 3' GAPDH amplicons in diluted buffy coat samples from cases 6 and 7 by 1-step real-time quantitative RT-PCR. Each sample, with a starting concentration of 10 ng/µL, was serially diluted in the following order: 10-fold (step 1), 5-fold (step 2), and 2-fold for 12 more steps (steps 3–14).

To further validate the observed data, we serially diluted 2 buffy coat RNA samples from healthy individuals (starting concentration, 10 ng/µL) and assessed the detection rates for the 5' and 3' GAPDH amplicons in these samples, using 1-step real-time quantitative RT-PCR. The results are expressed as copy number per microliter of total input RNA. As shown in panels B and C of Fig. 2Up , the 3' GAPDH amplicon was no longer detectable after only 5 consecutive dilution steps. On the other hand, the 5' amplicon was consistently detected in the more dilute samples, with up to 11 and 12 consecutive dilution steps for cases 6 and 7, respectively. The 5' and 3' GAPDH assays had shown equivalent sensitivities with the DNA calibrator, both detecting down to 48 copies/mL, thereby excluding the possibility of quantitative bias in this experiment. Collectively, these data supported the conclusion that 5' mRNA fragments constitute a majority of the GAPDH mRNA species in the circulation.

quantitative assessment of placental rna integrity in placental tissues
To explore the generality of the predominance of 5' mRNA fragments, we examined a panel of placenta-expressed transcripts. We compared the quantities of the 5', middle, and 3' regions of different mRNAs in placental tissues collected from 10 healthy pregnant women after caesarian section (gestational age, 38–40 weeks). Six placental transcripts, in addition to GAPDH mRNA, were chosen for this experiment, including ßhCG, PLAC1, TFPI2, ADM, INHBA, and PAPPA. The 5', middle, and 3' regions of each transcript were systematically quantified by 1-step real-time RT-PCR. Again the results were expressed as fractional mRNA concentrations relative to the 5' region to reflect the degree of RNA integrity of different transcripts in placental tissues (Table 1 ).


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  		Table 1. Comparison of the abundances of 5' and 3' mRNA fragments for different placenta-expressed transcripts in placental tissues and maternal plasma.

The 3 targeted amplicons for each of the 7 transcripts studied were detectable in all of the studied samples. As shown in Fig. 3 , except for PLAC1, the fractional mRNA concentrations differed significantly among the 3 targeted regions of each transcript (Friedman test, P <0.001). For GAPDH and ADM, pairwise multiple comparison showed a significant difference for every pair of targeted regions (Student–Newman–Keuls test, P <0.05). The mRNA concentration of the 5' GAPDH amplicon was 2.1-fold higher than that of the middle amplicon (middle-to-5' ratio = 0.47) and 34-fold higher than that of the 3' amplicon (3'-to-5' ratio = 0.029). Similarly, the mRNA concentration of the 5' ADM amplicon was 3.6-fold higher than that of the middle amplicon (middle-to-5' ratio = 0.28) and 6.7-fold higher than that of the 3' amplicon (3'-to-5' ratio = 0.15). For ßhCG, TFPI2, and INHBA, we observed a significant difference between the 5' and 3' amplicons (Student–Newman–Keuls test, P <0.05) and between the middle and 3' amplicons (Student–Newman–Keuls test, P <0.05). The 5' and middle amplicons, however, did not demonstrate any significant difference (Student–Newman–Keuls test, P >0.05). The median 3'-to-5' ratio was 0.62 for ßhCG, 0.071 for TFPI2, and 0.40 for INHBA. For the PAPPA gene, pairwise analysis also demonstrated a significant difference for every pair of targeted regions, but with a slightly different trend of expression across the 3 assays (Student–Newman–Keuls test, P <0.05). The mRNA concentration of the 5' PAPPA amplicon was 1.6-fold lower than that of the middle amplicon but 4.0-fold higher than that of the 3' amplicon (3'-to-5' ratio = 0.25). Lastly, for the PLAC1 gene, the fractional mRNA concentration gradually decreased as the target site was shifted from the 5' to the 3' end of the transcript, with the P value close to statistical significance (Friedman test, P = 0.067). These data suggested that, at the expression level, the placental tissue generally contained predominately the 5' fragments of placenta-expressed transcripts.



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  		Figure 3. Quantitative analysis of multiple regions of placenta-expressed transcripts in placental tissues.

Fractional mRNA concentrations with respect to the absolute concentrations of the 5' amplicon, as determined by 1-step real-time quantitative RT-PCR, are plotted on the y axis for the following genes: GAPDH, ADM, PLAC1, ßhCG, TFPI2, INHBA, and PAPPA. The lines inside the boxes denote the medians. The boxes represent the interval between the 25th and 75th percentiles, and the whiskers indicate the interval between the 10th and 90th percentiles. • denote outliers.

quantitative assessment of placental rna integrity in maternal plasma
We next investigated whether the placental RNA circulating in maternal plasma would also have an overrepresentation of the 5'-fragment region. Using 1-step real-time RT-PCR, we measured the abundance of the 5' and 3' ends of different placenta-expressed transcripts in the plasma of pregnant women. To facilitate placental RNA detection in maternal plasma (excluding GAPDH), our samples were chosen based on the expected time during pregnancy when the transcript is most highly expressed, as suggested by previous microarray data (9). Eight first-trimester samples (gestational age, 7–12 weeks) were assayed for ßhCG, 8 second-trimester samples (gestational age, 17–19 weeks) for GAPDH, 7 third-trimester samples for PLAC1 (gestational age, 37–40 weeks), TFPI2 (gestational age, 37–38 weeks), and PAPPA (gestational age, 37–41 weeks), and 10 third-trimester samples for ADM (gestational age, 37–40 weeks) and INHBA (gestational age, 37–40 weeks). The detection rates and absolute mRNA concentrations of the respective amplicons for each individual transcript were compared.

We observed a discrepancy in the rates of detection of the 5' and 3' amplicons for all of the studied transcripts (Table 2 ). Overall, the 5'-specific assays gave higher rates of placenta-derived RNA detection in maternal plasma compared with the 3'-specific assays, given that both types of assays had shown comparable sensitivities with the DNA calibrator during optimization. In terms of the absolute concentrations, we observed a significant decrease in the plasma mRNA concentration for GAPDH (Wilcoxon test, P <0.01), ßhCG (Wilcoxon test, P <0.01), TFPI2 (Wilcoxon test, P <0.05), ADM (Wilcoxon test, P <0.01), and INHBA (Wilcoxon test, P <0.01), as the target amplicon was changed from the 5' to the 3' end of the corresponding transcript (Fig. 4 ). The median 5' mRNA concentrations of GAPDH, ßhCG, TFPI2, ADM, and INHBA were 4274 (interquartile range, 2420–4813), 1031 (402–2216), 479 (321–868), 157 (66–263), and 39 (0–53) copies/mL, respectively. On the other hand, the median 3' mRNA concentrations were 114 (0–224), 156 (48–374), 130 (25–232), 0 (0–18), and 0 (0–0) copies/mL, respectively. Statistical comparisons between the maternal plasma concentrations of the 5' and 3' amplicons of PLAC1 or PAPPA were not performed because of their low detection rates in maternal plasma (Table 2 ). The 5' amplicons for both PLAC1 and PAPPA were detected in 29% (2 of 7), but the 3' amplicons were not detected in any of the tested samples. The plasma fractional mRNA concentrations (relative to the 5' region) of all transcripts are summarized in Table 1Up .


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  		Table 2. Detection rates of real-time RT-PCR assays targeting 5' and 3' regions of placenta-expressed transcripts in maternal plasma.



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  		Figure 4. Comparison of 5' and 3' regions of placenta-expressed transcripts in the plasma of pregnant women.

Plasma mRNA concentrations of 5' and 3' amplicons, as determined by 1-step real-time quantitative RT-PCR, are plotted on the y axis for the following genes: GAPDH, ßhCG, TFPI2, ADM, INHBA, PLAC1, and PAPPA. Both 5' and 3' assays showed similar detection limits for each transcript (12 copies/mL for ADM; 24 copies/mL for TFPI2, INHBA and PAPPA; and 48 copies/mL for GAPDH, ßhCG, and PLAC1). The lines inside the boxes denote the medians. The boxes represent the interval between the 25th and 75th percentiles, and the whiskers indicate the interval between the 10th and 90th percentiles. • denote outliers.

cross-gestational study of 5' and 3' ßHCG MRNA in maternal plasma
Apart from biological significance, our findings suggested that targeting the 5' end of a particular transcript could potentially improve the detection rate for RNA in maternal plasma. To illustrate such a clinical application, we compared the 5' and 3' ßhCG assays in maternal plasma from different gestation periods. We obtained plasma samples from 8 women in the first trimester of pregnancy (gestational age, 7–12 weeks) and from 17 women in the second trimester of pregnancy (gestational age, 16–20 weeks). As shown in Fig. 5 , the circulating ßhCG mRNA concentration displayed a decreasing trend with gestational age in both the 5' and 3' systems. Statistical analysis showed that the plasma mRNA concentrations of the 5' amplicon were significantly higher than those of the 3' amplicon for both trimesters (Wilcoxon test, P <0.01). As described above, in the first-trimester samples, the median concentrations of the 5' and 3' ßhCG amplicons were 1031 (interquartile range, 402-2216) and 156 (48-374) copies/mL, respectively. In the second-trimester samples, the median concentrations of the 5' and 3' ßhCG amplicons were 240 (0–363) and 0 (0–94) copies/mL, respectively. Furthermore, across gestations, the 5' ßhCG assay demonstrated a higher detection rate than the 3' ßhCG assay in maternal plasma. The detection rates of the 5' ßhCG assay were 100% for the first-trimester group and 53% for the second-trimester group, whereas the detection rates of the 3' ßhCG assay were only 75% for the first-trimester group and 29% for the second-trimester group. These data confirmed that the use of a 5'-specific system could enhance the sensitivity of mRNA detection in maternal plasma, suggesting potential implications in the future development of new assays for fetal RNA markers for noninvasive prenatal assessment.



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  		Figure 5. Cross-gestational analysis of the effect of amplicon location on the plasma concentrations of ßhCG mRNA in pregnant women.

Plasma mRNA concentrations of 5' and 3' ßhCG amplicons, as determined by 1-step real-time quantitative RT-PCR, are plotted against different stages of gestation. Both assays showed similar detection limits (48 copies/mL). The lines inside the boxes denote the medians. The boxes represent the interval between the 25th and 75th percentiles, and the whiskers indicate the interval between the 10th and 90th percentiles. • denote outliers.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
The discovery of placental RNA in the plasma of pregnant women has shown promise for noninvasive prenatal diagnosis and monitoring (8)(10)(11). However, little is currently known about the biology of circulating RNA. Recently it was demonstrated that cell-free RNA is surprisingly stable in plasma and serum (14). It has been postulated that cell-free circulating RNA is associated with particulate matter and thus protected from degradation by nucleases (15)(16). For a better understanding of the molecular characteristics of plasma RNA, we investigated the integrity of these molecular species.

We first studied the integrity of the GAPDH transcript in the plasma of healthy nonpregnant individuals by quantitatively comparing multiple regions (5', middle, and 3' regions) within the transcript in 1-step real-time RT-PCR assays. Our results suggested that circulating GAPDH mRNA population is predominantly 5' fragments rather than intact transcripts. We found that the fractional concentration of GAPDH mRNA (relative to the 5' region) decreased significantly as the amplicon position was moved away from the 5' end of the transcript and that the different target amplicons had significant correlations with one another. To exclude the possibility of analytical bias in the 1-step RT-PCR procedure, we performed 2-step RT-PCR analysis of 5' and 3' GAPDH amplicons in buffy coat RNA samples, using random and oligo(dT) primers in the reverse transcription step. Random priming produced results similar to those obtained by 1-step real-time analysis. In contrast, oligo(dT) priming gave a relatively higher absolute concentration of the 3' amplicon because RNA molecules without a 3' end could not be reverse transcribed in this system. To further validate our data, we used 1-step real-time RT-PCR to test the detection rates for 5' and 3' GAPDH amplicons in 2 sets of buffy coat RNA samples after serial dilutions. This experiment demonstrated the rapid disappearance of 3' fragments, in contrast to the persistent detection of 5' fragments. Given that we optimized both assays so that they were equally sensitive, the serial dilution experiment provided a physical means of demonstrating that there was indeed a higher abundance of transcript molecules with 5' ends than those with intact 3' ends. Collectively, these results confirmed the preponderance of 5' GAPDH mRNA fragments in the circulation.

To study whether the predominance of 5' mRNA fragments could be seen in other transcripts, we used the placenta as a model system for further investigation. Six different placental transcripts, in addition to GAPDH mRNA, were systematically analyzed for the 5' and 3' amplicons in the plasma of pregnant women. Five of the 7 genes (GAPDH, ßhCG, TFPI2, ADM, and INHBA) gave significantly higher absolute mRNA concentrations for the 5' amplicon than for the 3' amplicon. Moreover, for every gene under study (including PLAC1 and PAPPA), the 5' assay achieved a higher detection rate than the corresponding 3' assay in the analysis of plasma RNA. These findings provide direct evidence that circulating placental RNA is associated with a preponderance of 5' mRNA fragments in maternal plasma.

We observed a similar phenomenon when we studied the same panel of transcripts in placental tissues. Overall, the 7 genes could be classified into 3 categories with regard to the trend of expression from the analysis of the 5', middle, and 3' regions of each individual transcript. For GAPDH, ADM, and PLAC1, the fractional mRNA concentration (relative to the 5' amplicon) decreased gradually as the target site was changed from the 5' end to the middle part and from the middle part to the 3' end of the sequence. For ßhCG, TFPI2, and INHBA, we found no significant difference between the 5' and middle amplicons; however, the concentration of the 3' amplicon was significantly lower than the concentration of the 5' amplicon. Analysis of multiple regions of the PAPPA transcript showed yet another trend of expression in which the middle amplicon showed a significant increase across the 3 systems, with the 3' amplicon being relatively less abundant than the 5' amplicon. These results are consistent with previous reports (21)(22) documenting the detection of mRNA decay fragments in human and animal tissues.

To establish the biological relevance of this study, we addressed several important issues regarding experimental design. First, it is known that ßhCG is encoded by 6 highly similar genes (ßhCG1 through -3, -5, -7, and -8) clustered on chromosome 19 and is of high homology with the ß-subunit of the luteinizing hormone gene (ßLH) (23). To avoid introducing quantitative bias in the RT-PCR analysis, we ascertained that the 5', middle, and 3' primer sets for ßhCG were all complementary with the mRNA sequences arising from ßhCG3, -5, and -8, which are the major ßhCG mRNA species in the placenta at steady state (24). To minimize nonspecific amplification, we also incorporated in the primer and probe sequences several base mismatches to distinguish between ßhCG and ßLH. Second, for different RT-PCR systems to be compared on a valid ground, the choice of quantitative calibrators was another key aspect of this study. We constructed our own DNA calibrators by cloning all of the targeted sequences into the same plasmid for each studied transcript. Quantitative analysis of multiple regions within a transcript based on this type of calibrator would accurately reflect the relative changes in the measured mRNA concentrations and eliminate the potential bias from the use of multiple calibration curves. Optimization of assay sensitivity was also critical for accurate data interpretation in the analysis of plasma RNA. We obtained similar detection limits across the compared systems for every transcript under study, excluding the possibility of quantitative bias in our results. Theoretically, variable reverse transcription efficiency between different amplicons attributable to the differences in the primary genetic sequence may affect the quantification and thereby the comparison of concentrations among such amplicons. However, given the universal demonstration of the preponderance of 5' fragments in all of the 7 studied genes, it is unlikely that variation in the reverse transcription efficiency was a major confounding factor that was the sole basis of our observations.

In consideration of the origin of the observed 5' mRNA fragments in maternal plasma, we speculate that this biological phenomenon is likely to be associated with the mechanism of mRNA degradation. Previous studies have reported that the major pathway of mRNA decay in human cells proceeds from the 3' end of a transcript (25)(26)(27). One proposed model is that the human exosome, a protein complex consisting of 10 or more 3' exoribonucleases, interacts with an AU-rich element found in the 3'-untranslated region of an RNA transcript (26) through the binding of some AU-rich element–binding proteins (27). It has also been suggested that mRNA decay is correlated with the functional characteristics of human mRNAs (28). In this regard, the functional role, if any, of the observed placental RNA fragments in maternal plasma will require further investigation.

Apart from biological significance, our findings have potential clinical implications for the future study of placental RNA markers in maternal plasma. We have shown that, in addition to the quantitative differences, the detection rates of the 5' RT-PCR assays were higher than those of the corresponding 3' assays for detecting circulating placental RNA. Particularly, the plasma mRNAs of INHBA, PLAC1, and PAPPA could be detected only with the 5' amplicon. In addition, in the cross-gestational study, the 5' ßhCG system could detect its target in 100% of first-trimester samples and 53% of second-trimester samples. On the other hand, the 3' ßhCG system was positive for only 75% and 29%, respectively, of the corresponding samples. Of note, a recent study reported another ßhCG assay that amplified the middle region of the mRNA sequence and detected ßhCG mRNA in 100% of first-trimester samples and 42% of second-trimester samples (8). Collectively, these data have suggested that targeting the 5' end of a transcript could potentially increase the performance of a placental RNA marker in maternal plasma for noninvasive prenatal diagnosis. Whether the degraded transcripts detected in maternal plasma could be representative of the pathophysiology of pregnancy-associated diseases is beyond the scope of this study and remains to be investigated.

In conclusion, we have demonstrated that circulating placental RNA is associated with a preponderance of 5' mRNA fragments in maternal plasma. In addition to the biological relevance, the findings of our study have particular implications for the future development of fetal RNA markers for noninvasive prenatal diagnosis. For example, this information could be used to improve the rate of placental RNA detection in maternal plasma, thus facilitating the establishment of more molecular markers for pregnancy-associated diseases, including preeclampsia and fetal chromosomal aneuploidies. Our findings may also have implications for other clinical uses of plasma RNA, particularly in the detection of tumor RNA for cancer diagnosis (29).


   Acknowledgments
 
We thank Dr. Lisa Y.S. Chan, Katherine C.K. Chow, and Nicole Y.L. Lam for technical assistance. We are also grateful to Dr. Enders K.O. Ng for helpful discussion and advice at the start of the project. This work was supported by a Central Allocation Grant (CUHK 01/03C) from the Research Grants Council of the Hong Kong Special Administrative Region (China). Several of the co-authors hold patents or patent applications on aspects of circulating nucleic acids in plasma.


   Footnotes
 
1 Nonstandard abbreviations: RT-PCR, reverse transcription-PCR; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; ßhCG, ß-subunit of human chorionic gonadotropin; PLAC1, placenta-specific 1; TFPI2, tissue factor pathway inhibitor 2; ADM, adrenomedullin; INHBA, inhibin-ß A subunit; and PAPPA, pregnancy-associated plasma protein A.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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