Exploring Optimal Biomarker Sources: A Comparative Analysis of Exosomes and Whole Plasma in Fasting and Non-Fasting Conditions for Liquid Biopsy Applications.
Author(s): Nasu M, Khadka VS, Jijiwa M, Kobayashi K, Deng Y
Publication: Int J Mol Sci, 2023, Vol. 25, Page
PubMed ID: 38203541 PubMed Review Paper? No
Purpose of Paper
This paper compared the small non-coding RNA profile and metabolome of plasma and exosomes isolated from blood collected from fasting and non-fasting volunteers.
Conclusion of Paper
A total of 2,425 microRNAs (miRNAs), 92 Piwi-interacting RNAs (piRNAs), 252 transfer RNAs (tRNAs), and 96 small RNAs of other types were detected in plasma specimens; 2,400 miRNAs, 68 piRNAs, 233 tRNAs, and 85 small RNAs of other types were detected in exosomes. The RNA profiles of fasting and non-fasting volunteers were very strongly correlated for plasma (r=0.9868) and exosomes (r=0.9994); and, for each subtype of small RNA, the correlation between specimens from fasting and non-fasting volunteers was higher when exosomes were analyzed rather than plasma. There were 810 miRNAs, 30 piRNAs, and 59 snoRNA that were differentially expressed between plasma and exosomes, including higher levels of many platelet- and/or erythrocyte-expressed miRNAs in plasma than exosomes.
Only 261 of the 513 metabolites found in plasma were also detected in exosomes. Levels of metabolites were very strongly correlated between fasting and non-fasting volunteers for plasma (r-0.9634) and exosomes (r=0.9435), but fasting status had a larger effect on metabolite levels in exosomes than plasma. Fasting status affected (≥1.4-fold difference) levels of 37 lipids, 71 triglycerides/diglycerides, 26 fatty acids, 2 amino acids, and 17 bile acids in plasma, but only levels of 5 lipids were affected by fasting status when exosomes were analyzed.
Studies
-
Study Purpose
This study compared the small non-coding RNA profile and metabolome of plasma and exosomes isolated from blood collected from fasting and non-fasting volunteers. Blood was collected from four volunteers (males: 30, 38, and 52 years; female: 52 years) into K2EDTA Vacutainer tubes after an overnight fast and again 3 and 5 h after breakfast. Plasma was separated by centrifugation at 1500 g for 15 min. Exosomes were isolated from plasma using the Capturem Extracellular Vesicle Isolation Kit. RNA was isolated from plasma and exosomes using the miRNeasy Serum/Plasma Kit. Sequencing libraries were prepared with the QIAseq miRNA Library Kit and QIAseq miRNA NGS 12 Index IL and sequenced on a NextSeq 500 instrument. Lipid and metabolite profiles were analyzed by ultra-performance liquid chromatography (UPLC) tandem mass spectrometry (MS).
Summary of Findings:
A total of 2,425 microRNAs (miRNAs), 92 Piwi-interacting RNAs (piRNAs), 252 transfer RNAs (tRNAs), and 96 small RNAs of other types were detected in plasma specimens, and 2,400 miRNAs, 68 piRNAs, 233 tRNAs, and 85 small RNAs of other types were detected in exosomes. The RNA profiles were very strongly correlated between fasting and non-fasting volunteers for both plasma (r=0.9868) and exosomes (r=0.9994). For exosomes, the correlation in miRNA levels was perfect between specimens obtained from fasting and non-fasting volunteers (R=1), and the correlation between levels of each subtype of small RNA was higher when exosomes were used rather than whole plasma. In principal component analysis, there was complete overlap between the fasting and non-fasting specimens from each of the four volunteers based on the exosomal RNA profile. In contrast, principal component analysis based on the plasma RNA profile, did not cluster specimens by either fasting status or patient source. There were significant differences in the expression of 810 miRNAs between plasma and exosomes (P<0.05). Seven of the most upregulated miRNAs in plasma are reported to be from blood. Further analysis of 28 miRNAs known to be expressed in platelets and/or erythrocytes revealed that the majority (25 of 28 with P<0.01, another P=0.0124) were expressed at higher levels in plasma than exosomes. The authors state that these blood-cell derived miRNAs introduce bias when analyzing plasma rather than exosomes. A total of 30 piRNAs and 59 snoRNA were differentially expressed between plasma and exosomes.
Only 261 of the 513 metabolites found in plasma were detected in exosomes. Levels of metabolites were very strongly correlated between fasting and non-fasting volunteers for both plasma (r-0.9634) and exosomes (r=0.9435). Fasting status had a larger effect on metabolite levels in exosomes than plasma. In plasma specimens, the correlation between specimens from fasting and non-fasting volunteers was >0.99 for lipids, amino acids, and fatty acids but only 0.8936 for bile acids. Among amino acids, alanine levels were slightly higher in plasma from non-fasting than fasting volunteers (P=0.0027), but levels of 28 other amino acids in plasma were not affected by fasting status. Levels of ten bile acids were higher and seven were lower in plasma from non-fasting than fasting volunteers. The majority of fatty acids (24 of 39) had lower levels in plasma from non-fasting volunteers than fasting volunteers, but caprylic acid (C8:0) and hexanoic acid (C6:0) were both significantly higher in plasma from non-fasting volunteers than fasting volunteers (P=0.0046 and P=0.014, respectively). A total of 37 lipids were differentially expressed between plasma from fasting and non-fasting volunteers. In contrast 5 lipids, but no fatty acids, amino acids, or bile acids, were affected by fasting status when exosomes were analyzed.
Biospecimens
Preservative Types
- None (Fresh)
Diagnoses:
- Normal
Platform:
Analyte Technology Platform Lipid HPLC-MS RNA Next generation sequencing Steroid LC-MS or LC-MS/MS Small molecule HPLC-MS Pre-analytical Factors:
Classification Pre-analytical Factor Value(s) Biospecimen Acquisition Time of biospecimen collection Fasting
3 or 5 h after eating
Biospecimen Aliquots and Components Blood and blood products Plasma exosomes
Plasma