Abstract
Development of maternal blood transcriptomic markers to monitor placental function and risk of obstetrical complications throughout pregnancy requires accurate quantification of gene expression. Herein, we benchmark three state-of-the-art expression profiling techniques to assess in maternal circulation the expression of cell type-specific gene sets previously discovered by single-cell genomics studies of the placenta. We compared Affymetrix Human Transcriptome Arrays, Illumina RNA-Seq, and sequencing-based targeted expression profiling (DriverMapTM) to assess transcriptomic changes with gestational age and labor status at term, and tested 86 candidate genes by qRT-PCR. DriverMap identified twice as many significant genes (q < 0.1) than RNA-Seq and five times more than microarrays. The gap in the number of significant genes remained when testing only protein-coding genes detected by all platforms. qRT-PCR validation statistics (PPV and AUC) were high and similar among platforms, yet dynamic ranges were higher for sequencing based platforms than microarrays. DriverMap provided the strongest evidence for the association of B-cell and T-cell gene signatures with gestational age, while the T-cell expression was increased with spontaneous labor at term according to all three platforms. We concluded that sequencing-based techniques are more suitable to quantify whole-blood gene expression compared to microarrays, as they have an expanded dynamic range and identify more true positives. Targeted expression profiling achieved higher coverage of protein-coding genes with fewer total sequenced reads, and it is especially suited to track cell type-specific signatures discovered in the placenta. The T-cell gene expression signature was increased in women who underwent spontaneous labor at term, mimicking immunological processes at the maternal-fetal interface and placenta.
Highlights
IntroductionCellular and cell-free RNAs in blood that originate from (or are specific to) the primary tumor or organ of interest are especially sought as candidate biomarkers[2,7,8] and, more recently, owing to advances in single-cell genomics[9], researchers developed cell type-specific signatures of tissues, e.g., the placenta
Since organ and/or cell type-specific transcripts are expected to have low expression in whole blood, it is essential that quantification of RNA abundance is accurate enough so that modest, and eventually coordinated, gene expression changes can be leveraged as biomarkers that have clinical utility
RRNA depletion and globin reduction have been shown to mitigate some of these issues, they require a large amount of total RNA and may induce biases in the quantification of gene expression[11]
Summary
Cellular and cell-free RNAs in blood that originate from (or are specific to) the primary tumor or organ of interest are especially sought as candidate biomarkers[2,7,8] and, more recently, owing to advances in single-cell genomics[9], researchers developed cell type-specific signatures of tissues, e.g., the placenta This approach holds the promise to unravel the complexity of the maternal-fetal molecular dialogue[2] and to aid in developing liquid biopsies for prediction of the ‘great obstetrical syndromes’[10]. We have for the first time evaluated the ability of these high-throughput methods to quantify in maternal whole blood the expression of cell type-specific signatures derived from single-cell genomics of the placenta, and we have determined whether these signatures are indicative of the onset of term parturition
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