Extracellular vesicles are selectively enriched in RNA that has potential as disease biomarkers. To systemically characterize circulating extracellular RNA (exRNA) profiles, researchers from the Medical College of Wisconsin performed RNA sequencing analysis on plasma extracellular vesicles derived from 50 healthy individuals and 142 cancer patients. Of ~12.6 million raw reads for each individual, the number of mappable reads aligned to RNA references was ~5.4 million including miRNAs (~40.4%), piwiRNAs (~40.0%), pseudo-genes (~3.7%), lncRNAs (~2.4%), tRNAs (~2.1%), and mRNAs (~2.1%). By expression stability testing, they identified a set of miRNAs showing relatively consistent expression, which may serve as reference control for exRNA quantification. By performing multivariate analysis of covariance, they identified significant associations of these exRNAs with age, sex and different types of cancers. In particular, down-regulation of miR-125a-5p and miR-1343-3p showed an association with all cancer types tested (false discovery rate <0.05). The researchers developed multivariate statistical models to predict cancer status with an area under the curve from 0.68 to 0.92 depending cancer type and staging. This is the largest RNA-seq study to date for profiling exRNA species, which has not only provided a baseline reference profile for circulating exRNA, but also revealed a set of RNA candidates for reference controls and disease biomarkers.
Statistical summary of RNA species detected by RNA-seq across 192 libraries.
(A) Percentage of each RNA species in all mapped RNAs. (B) Number of mapped unique RNA references in different RNA species. (C) Percentage (sorted from high to low) of each detected mature miRNAs in all mapped miRNA reads. The top 32 miRNAs (quantile 0.98) are highlighted in red and are also shown in embedded graph. (D) Percentage (sorted from high to low) of each detected piwiRNAs in all mapped reads. Top 10 piwiRNAs (quantile 0.98) are highlighted in red and also shown in embedded graph. RNA transcripts with >4 RPM were used. (E) The top 32 miRNAs and their isoform proportions. The color bars represent the percentage of each miRNA isoform.