Abstract
Recent studies demonstrate that small extracellular vesicles (sEVs), which are found in all biofluids, play critical roles in intercellular communication by channeling proteins, DNA, and RNAs. MicroRNAs (miRNAs) that are packaged in sEVs have emerged as critical deliverable regulators in recipient cells. Since sEVs secreted by normal and diseased cells carry different miRNA cargos, recent sEV-miRNA profiling studies suggest that they may help identify novel circulating biomarkers. However, cell/disease-specific sEVs circulating in diverse biofluids, once isolated, provide low miRNA quantities, which are generally difficult to quantify using conventional spectrometric methodologies. Small non-coding RNA Next Generation Sequencing (NGS), which allows for the amplification of cloned miRNA sequences, offers a valuable opportunity to evaluate the miRNA cargos of sEVs. Unfortunately, commercial cDNA library preparation procedures often require RNA inputs well above the unquantifiable amounts available from isolated sEVs. Thus, considering the robustness and multiplexing capabilities of our existing cDNA library preparation procedure (i.e., initially optimized for the analysis of low-input, highly degraded, formalin-fixed paraffin-embedded (FFPE) RNA), we sought to evaluate its applicability for the analysis of sEV miRNAs. Importantly, taking into account the recent technical clustering improvements of sequencing chips, we sought to adapt our transcript barcoding approach within a paired-end, dual index-compatible cDNA library preparation workflow to enhance our sequencing and multiplexing capabilities. Using RNA extracted from 8.4 × 10(9) sEVs in 16 replicates, and from decreasing amounts of sEVs from 10(10) sEVs to as low as 2.5 × 10(7) sEVs, we evaluated the reproducibility and sensitivity of this methodology. The data demonstrate that the 16 3' adenylated DNA barcodes allow for highly reproducible and sensitive detection of sEV-miRNA profiles across repeats using as low as 3.15 pg of total small non-coding RNAs or 1.35 pg of miRNAs.