Spearman correlation for the expression level of detected microRNAs.

<p>While different numbers of miRNAs were quantified between duplicated samples, the majority of these miRNA were commonly observed. The expression level of these commonly observed miRNA were highly correlated for both duplicated samples (r>0.9) and across samples from different subjects (r>0.74).</p

Venn diagram of mature miRNA species detected in each treatment group.

<p>Plasma samples libraries from a single health donor generated using no PK, PK treatment before GITC, PK treatment in GITC show strong concordance of miRNA species detected, with PK treatment in GITC method showing higher sensitivity than others.</p

Top 20 miRNA by read counts in duplicated control samples.

<p>Top 20 miRNA by read counts in duplicated control samples.</p

High Throughput Sequencing of Extracellular RNA from Human Plasma

<div><p>The presence and relative stability of extracellular RNAs (exRNAs) in biofluids has led to an emerging recognition of their promise as ‘liquid biopsies’ for diseases. Most prior studies on discovery of exRNAs as disease-specific biomarkers have focused on microRNAs (miRNAs) using technologies such as qRT-PCR and microarrays. The recent application of next-generation sequencing to discovery of exRNA biomarkers has revealed the presence of potential novel miRNAs as well as other RNA species such as tRNAs, snoRNAs, piRNAs and lncRNAs in biofluids. At the same time, the use of RNA sequencing for biofluids poses unique challenges, including low amounts of input RNAs, the presence of exRNAs in different compartments with varying degrees of vulnerability to isolation techniques, and the high abundance of specific RNA species (thereby limiting the sensitivity of detection of less abundant species). Moreover, discovery in human diseases often relies on archival biospecimens of varying age and limiting amounts of samples. In this study, we have tested RNA isolation methods to optimize profiling exRNAs by RNA sequencing in individuals without any known diseases. Our findings are consistent with other recent studies that detect microRNAs and ribosomal RNAs as the major exRNA species in plasma. Similar to other recent studies, we found that the landscape of biofluid microRNA transcriptome is dominated by several abundant microRNAs that appear to comprise conserved extracellular miRNAs. There is reasonable correlation of sets of conserved miRNAs across biological replicates, and even across other data sets obtained at different investigative sites. Conversely, the detection of less abundant miRNAs is far more dependent on the exact methodology of RNA isolation and profiling. This study highlights the challenges in detecting and quantifying less abundant plasma miRNAs in health and disease using RNA sequencing platforms.</p></div

Read counts in all samples analyzed.

<p>Read counts in all samples analyzed.</p

Top expressing miRNA with target genes and reported dysregulation in human disease.

<p>Top expressing miRNA with target genes and reported dysregulation in human disease.</p

Schematic summarizing plasma sample treatments used in this study.

<p>RNA isolation was performed with or without proteinase K treatment and ribodepletion on fresh or archived samples.</p