Comparing small urinary extracellular vesicle purification methods with a view to RNA sequencing—Enabling robust and non-invasive biomarker research

Abstract

Small extracellular vesicles (EVs) are 50–200 nm sized mediators in intercellular communication that reflect both physiological and pathophysiological changes of their parental cells. Thus, EVs hold great potential for biomarker detection. However, reliable purification methods for the downstream screening of the microRNA (miRNA) cargo carried within urinary EVs by small RNA sequencing have yet to be established. To address this knowledge gap, RNA extracted from human urinary EVs obtained by five different urinary EV purification methods (spin column chromatography, immunoaffinity, membrane affinity, precipitation and ultracentrifugation combined with density gradient) was analyzed by small RNA sequencing. Urinary EVs were further characterized by nanoparticle tracking analysis, Western blot analysis and transmission electron microscopy. Comprehensive EV characterization established significant method-dependent differences in size and concentration as well as variances in protein composition of isolated vesicles. Even though all purification methods captured enough total RNA to allow small RNA sequencing, method-dependent differences were also observed with respect to library sizes, mapping distributions, number of miRNA reads and diversity of transcripts. Whereas EVs obtained by immunoaffinity yielded the purest subset of small EVs, highly comparable with results attained by ultracentrifugation combined with density gradient, precipitation and membrane affinity, sample purification by spin column chromatography indicated a tendency to isolate different subtypes of small EVs, which might also carry a distinct subset of miRNAs. Based on our results, different EV purification methods seem to preferentially isolate different subtypes of EVs with varying efficiencies. As a consequence, sequencing experiments and resulting miRNA profiles were also affected. Hence, the selection of a specific EV isolation method has to satisfy the respective research question and should be well considered. In strict adherence with the MISEV (minimal information for studies of extracellular vesicles) guidelines, the importance of a combined evaluation of biophysical and proteomic EV characteristics alongside transcriptomic results was clearly demonstrated in this present study.

Comparison of urinary extracellular vesicle isolation methods for transcriptomic biomarker research in diabetic kidney disease

“…While this aspect has not been widely shown in earlier studies, this finding is in agreement with the results published by Mussak et al. (Mussack et al., 2019). The absence of EV markers in NG samples can be partly explained by the low yield of vesicles in this protocol as measured by NTA.…”

“…Therefore, the NG samples were measured near the lower end of the range and it is possible that NG yield values were overestimated. Lower RNA yields from NG compared to other isolation methods has been reported by previous studies (Mussack et al, 2019;Wachalska et al, 2016).…”

“…The total miRNA raw counts from UC samples were higher than for HFD and NG. However, miRNA counts from HFD and NG are in the range of counts reported in other uEV small RNA sequencing studies (Mussack et al, 2019). Identified miRNAs (> 5 normalized counts in all samples within a method) or top ten identified miRNAs showed to be around 90% similar between isolation methods.…”

“…Few systematic studies have compared uEV isolation methods for downstream effects on RNA transcriptomics results (Cheng et al., 2014; Mussack et al., 2019; Park et al., 2020; Srinivasan et al., 2019). However, these published studies have focused mainly in small/miRNA sequencing and most of them have used only urine from healthy donors, which may lead to misinterpretations of performance.…”

Diabetic Nephropathy: Perspective on Extracellular Vesicles

“…EVs obtained by immuneaffinity yielded the purest subset of small EVs, while purification by spin column chromatography indicated a tendency to isolate different subtypes of small EVs. Indeed different EV purification methods could isolate different subtypes of EVs with varying efficiencies, thus influencing the results of miRNA profiles (27). Isolating a pure vesicle of a subpopulation is relatively difficult because of the size overlap in the vesicle subpopulations.…”