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. 2022 Jul 18;45(4):621–638. doi: 10.1007/s13402-022-00688-3

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© The Author(s) 2022

Open AccessThis article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/.

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Fig. 1 — Establishment and characterization of standardized procedure for circulating small EV isolation. Small RNA sequencing was used to compare different plasma preparation methods of purifying circulating EV-miRs. RNAs were isolated by using two types of plasma small EVs preparation methods. (A) EV and corresponding plasma samples were prepared from two mCRC patient specimens (patients 10 and 11). Western blot was performed to analyze the small EV protein markers including CD9 and Syntenin-1, and a negative marker of cellular contamination, Calnexin. (B) NanoSight data of microvesicles eluted from the membrane affinity column. (C) Scanning electron microscopy analysis of CRC patients’ circulating small EVs. (D-E) RNA size distribution for fresh (D) and hemolytic (E) blood samples. (F) Small RNA-sequencing experiments were performed. Upon read alignment, miRNA expression levels were determined based on the normalized read count values. PCA plots are shown to depict the distributions of miRNA expression profiles in fresh (blue) vs. frozen (red) plasma samples. Significant separation of the two groups is indicative of distinct transcriptome signatures