Unique extracellular RNA profiles in different fractions of plasma

Circulating extracellular RNAs (exRNAs) have great promise as novel clinically plasma-based biomarkers for cancer diagnosis and prognosis. knowledge of the difference or association between these different sources of exRNAs is very limited. We performed a sequential physical and biochemical precipitation to receive 4 plasma fractions (platelets and cell debris, Thrombin-treated fraction, extracellular vesicles and supernatant) from each plasma sample. After total RNA extraction, we made ligation-free libraries and performed RNA-seq to evaluate full spectrum of RNA abundance in each fraction. All RNAs were included without size selection during the library preparation. We utilized a successive stepwise alignment strategy to map the RNA sequences to different RNA categories. We found that each plasma fraction had its own unique distribution of RNA species. Hierarchical cluster analysis showed similarity in samples with same fraction and significant differences between different fractions. In addition, we also observed abundance difference of unique transcript. Furthermore, a considerable proportion of exogenous RNAs and novel predicted miRNAs in all plasma fractions were detected. These results demonstrate that thorough inspection of all plasma fractions is necessary for exRNA-based biomarker study and appropriate sampling processes is needed for illustrating the complexity of plasma-based EVs study. Overall design: Total RNA extracted from each of the 4 fractions of 10 plasma samples. Sequencing data of all RNA species was generated by Illumina Hiseq sequencer.