Extracellular circular RNA profiles in plasma and urine of healthy, male college athletes

Abstract

Circular RNA (circRNA) are a recently discovered class of RNA characterized by a covalently-bonded back-splice junction. As circRNAs are inherently more stable than other RNA species, they may be detected extracellularly in peripheral biofluids and provide novel biomarkers. While circRNA have been identified previously in peripheral biofluids, there are few datasets for circRNA junctions from healthy controls. We collected 134 plasma and 114 urine samples from 54 healthy, male college athlete volunteers, and used RNASeq to determine circRNA content. The intersection of six bioinformatic tools identified 965 high-confidence, characteristic circRNA junctions in plasma and 72 in urine. Highly-expressed circRNA junctions were validated by qRT-PCR. Longitudinal samples were collected from a subset, demonstrating circRNA expression was stable over time. Lastly, the ratio of circular to linear transcripts was higher in plasma than urine. This study provides a valuable resource for characterization of circRNA in plasma and urine from healthy volunteers, one that can be developed and reassessed as researchers probe the circRNA contents of biofluids across physiological changes and disease states.

Fig. 1

Study Workflow.

Fig. 2

CircRNAs were predicted from 134 plasma (a,b) and 114 urine (c,d) samples using 6 different bioinformatic tools. 965 circRNA were identified by all 6 tools in plasma (a; red bar), and 72 circRNA were identified by all 6 tools in urine (c; red bar). Genomic features located within predicted back-spliced junctions in plasma (b) and urine (d), respectively.

Fig. 3

(a,b) Highly-expressed, predicted back-spliced junctions were validated by qRT-PCR. qRT-PCR validation of the 15 most highly expressed circRNA found in plasma (a) and urine (b), respectively. Each circRNA was examined in 10 cDNA samples from the same source RNA as sequenced samples. (c,d) Circular-linear ratios are higher in plasma than urine. Linear splice junction expression plotted against circular splice junction expression in plasma (c) and urine (d). Points representing circRNA between 1-fold and 5-fold higher than their linear counterparts are blue; 5x or higher are red.

Fig. 4

Participants sequenced five or more times have less inter-sample variation. CircRNA populations identified in plasma (a,c) and urine (b,d) from participants sampled five or more times. (a,b) Heatmaps showing the log-normalized JRPM expression of plasma (a) and urine (b) samples taken longitudinally from the same participant. The coefficient of variation (CV) of circRNA expression is significantly lower across individual participant samples when compared to the entire dataset (c, plasma; and d, urine). ****p <  = 0.0001.