High Throughput Sequencing of Extracellular RNA from Human Plasma

Abstract

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.

Evaluation of serum extracellular vesicle isolation methods for profiling miRNAs by next‐generation sequencing

“…This holds true particularly for extracellular RNA, which bears additional challenges such as low concentrations, diminished RNA integrity and high variability between individuals. Indeed, recent publications have highlighted the impact of cell-free RNA extraction strategies on small RNA-Seq, reporting quantitative and qualitative differences in resulting sequencing libraries [6,7]. …”

Phospho‐RNA‐seq: a modified small RNA‐seq method that reveals circulating mRNA and lncRNA fragments as potential biomarkers in human plasma

“…These efforts have begun to elucidate the complex composition of exRNA in blood (Freedman et al, 2016;Yeri et al, 2017;Godoy et al, 2018;Max et al, 2018). There have been indications of extracellular mRNA and lncRNA in some studies of plasma, but results have been inconsistent, with some profiling studies reporting a variable percentage of them and others not even reporting their presence (Huang et al, 2013;Koh et al, 2014;Freedman et al, 2016;Yuan et al, 2016;Danielson et al, 2017;Yeri et al, 2017;Godoy et al, 2018;Max et al, 2018). Moreover, these profiling studies have used a variety of methods to evaluate exRNA (e.g., microarrays and different methodologies for RNA-seq) which, not surprisingly, contributes to the variation in findings.…”

Comprehensive Profiling of the Circulatory miRNAome Response to a High Protein Diet in Elderly Men: A Potential Role in Inflammatory Response Modulation

“…Contrastingly, HTS has the advantage of using total read count rather than a few selected transcripts for normalization, yet this depends on the total efficiency of extraction . Additionally, qPCR abundance of miR‐100‐5p and ‐203a showed low expression and it has been reported that high sequencing depth is required to detect lowly expressed genes . Thus, it is possible that in the case of these low abundance miRNAs, sequencing at a depth of at least 20 million reads per library provided greater sensitivity than the qPCR technique used.…”

“…[76] Additionally, qPCR abundance of miR-100-5p and -203a showed low expression and it has been reported that high sequencing depth is required to detect lowly expressed genes. [77] Thus, it is possible that in the case of these low abundance miRNAs, sequencing at a depth of at least 20 million reads per library provided greater sensitivity than the qPCR technique used. It would have been ideal to further validate the mRNA expression findings at the level of the post-transcriptionally regulated target proteins; however, due to insufficient remaining PBMCs this was not possible.…”