The authors then analyzed cfRNA from 204 plasma samples collected from 60 pregnant women during the first, second, and third trimesters, as well as on the first postpartum day.
Cluster 2 included placenta-derived genes only found during pregnancy, which had previously been reported by the authors in plasma cfRNA (8).
Overall, composition of microbial plasma cfRNA remained stable over the course of pregnancy.
In light of the great potential of cfRNA analysis, it is important to keep in mind that its main advantage (easy accessibility of RNA shed anywhere in the body) also represents a potential weakness.
The cfRNA was extracted from 1 mL of plasma using a Plasma/Serum Circulating RNA and Exosomal Purification kit (Norgen, cat 42800).
We first evaluated 4 methods (ScriptSeq, RNase H, Exome, and Clontech) for preparing sequencing libraries from cfRNA. The ScriptSeq method uses random primers to synthesize cDNA from total cfRNA (Illumina ScriptSeq v2 kit).
Reads in the cfRNA libraries that failed to align to human transcriptome were extracted and aligned to the bacteriophage [phi] X 174, which is used as a control sequence in the Illumina platform.
In this study, each patient had multiple cfRNA samples collected from different time points.
cfRNA concentrations in seminal plasma from the 10 healthy individuals varied from 0.87 mg/L to 3.64 mg/L with a mean (SD) of 1.75 mg/L (0.92 mg/L).
Such an analysis has previously been done in studies of cfRNA integrity in serum and saliva (21, 23, 24).
The high stability of cfsRNA, like that of cfRNA in other body fluids, may partially be a consequence of binding with complex organic molecules.
 Nonstandard abbreviations: cfRNA, cell-free RNA; cfsRNA, cell-free seminal RNA; RT-PCR, reverse-transcription PCR; RT-qPCR, reverse-transcription quantitative real-time PCR; SNK, Student-Newman-Keuls.