Small RNA Sequencing across Diverse Biofluids Identifies Optimal Methods for exRNA Isolation

Abstract

Graphical AbstractHighlights d exRNA sequencing complexity and reproducibility varies across isolation methods d Deconvolution shows differential access to exRNA carriers by different methods d Performance of exRNA isolation methods vary across biofluids and RNA species d miRDaR enables customized selection of optimal exRNA isolation methods SUMMARYPoor reproducibility within and across studies arising from lack of knowledge regarding the performance of extracellular RNA (exRNA) isolation methods has hindered progress in the exRNA field. A systematic comparison of 10 exRNA isolation methods across 5 biofluids revealed marked differences in the complexity and reproducibility of the resulting small RNA-seq profiles. The relative efficiency with which each method accessed different exRNA carrier subclasses was determined by estimating the proportions of extracellular vesicle (EV)-, ribonucleoprotein (RNP)-, and highdensity lipoprotein (HDL)-specific miRNA signatures in each profile. An interactive web-based application (miRDaR) was developed to help investigators select the optimal exRNA isolation method for their studies. miRDar provides comparative statistics for all expressed miRNAs or a selected subset of miRNAs in the desired biofluid for each exRNA isolation method and returns a ranked list of exRNA isolation methods prioritized by complexity, expression level, and repro-ducibility. These results will improve reproducibility and stimulate further progress in exRNA biomarker development.

Comparison of miRNA profiling methods using synthetic miRNA pools and standardized exRNA samples reveals substantial performance differences

“…For isolated RNA, the median CV was higher with small RNA-sequencing (33.4%) than with EdgeSeq (14.4%). As previously reported for RNA-seq (Srinivasan et al, 2019), CVs decreased as signal increased, but overall CVs remained higher for the plasma RNA samples than for the synthetic pools.…”

“…Signal intensities between data generated from isolated RNA versus crude plasma were moderately well correlated for EdgeSeq (R = 0.62, Supplemental Figure 6). To assess whether the use of crude biofluid biased against miRNAs carried in certain subcompartments in plasma, we inspected signals for miRNAs previously found to be differentially associated with 5 different subcompartments in human serum: CD63+ EVs, CD81/CD9+ EVs, AGO2+ RNPs, HDL, and lipoprotein-free fraction (LFF) (Srinivasan et al, 2019). We found no obvious systematic differences in signal according to the assigned subcompartment (Supplemental Figure 6), suggesting that EdgeSeq is capable of detecting miRNAs that are preferentially associated with each subcompartment within crude plasma samples.…”

Charting extracellular transcriptomes in The Human Biofluid RNA Atlas

“…In the discovery cohort, 20 different biofluids were collected in two donors or in a pool of [4][5] donors. In the case/control cohorts, selected biofluids (sputum, CSF, urine and saliva) were collected in [8][9][10][11][12] patients and an equal number of healthy controls. Both small RNA sequencing and mRNA capture sequencing were performed in the discovery cohort.…”

“…To date, the characterization of exRNAs in biofluids has mainly focused on small RNAs in blood derived samples [1][2][3][4][7][8][9][10] . MicroRNA (miRNA) is the most studied small RNA biotype in biofluids, but other small RNAs, such as piwi-interacting RNAs (piRNAs), small nuclear RNAs (snRNAs), small nucleolar RNAs (snoRNAs), ribosomal RNAs (rRNAs), transfer RNA fragments (tRNAs) and Y-RNAs have also been identified 5,6,8,[10][11][12] . While most studies have focused on plasma and serum, some groups have investigated the small RNA content of other human biofluids.…”

“…Weber et al 12 was the first to compare the miRNA content in 12 different human biofluids 5,8,10,10,11 . The distribution of small RNA biotypes clearly varies across these biofluids, with a high abundance of piRNAs and tRNAs reported in urine and a high abundance of Y-RNAs in plasma 6,8,11 .…”