Comparison of Serum Exosome Isolation Methods on Co-precipitated Free MicroRNAs

Cheng, Yirui; Qu, Xiangyun; Dong, Zhaonan; Zeng, Qingyu; Ma, Xin; Jia, Yunli; Li, Ruochen; Jiang, Xiaoxu; Williams, Cecilia; Wang, Tao; Xia, Weiliang

doi:10.21203/rs.2.18614/v1

Cited by 5 publications

(6 citation statements)

References 29 publications

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“…When isolating EVs from plasma, these samples show a higher amount of small particles, presumably lipoproteins, and a likely co-enrichment of free miRNA (Cheng et al, 2020), as seen by the EM, NTA, and Bioanalyzer RNA data. Thus, for plasma the preferred choice of method is a bit different, here Izon  isolates the most EV proteins, but not the purest, as it identified most EV proteins but also the most non-EV proteins.…”

Section:  Discussionmentioning

confidence: 86%

Molecular evaluation of five different isolation methods for extracellular vesicles reveals different clinical applicability and subcellular origin

Veerman

Teeuwen

Czarnewski

et al. 2021

J of Extracellular Vesicle

197

150

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Extracellular vesicles (EVs) are increasingly tested as therapeutic vehicles and biomarkers, but still EV subtypes are not fully characterised. To isolate EVs with few co-isolated entities, a combination of methods is needed. However, this is timeconsuming and requires large sample volumes, often not feasible in most clinical studies or in studies where small sample volumes are available. Therefore, we compared EVs rendered by five commonly used methods based on different principles from conditioned cell medium and 250 µl or 3 ml plasma, that is, precipitation (Exo-Quick ULTRA), membrane affinity (exoEasy Maxi Kit), size-exclusion chromatography (qEVoriginal), iodixanol gradient (OptiPrep), and phosphatidylserine affinity (MagCapture). EVs were characterised by electron microscopy, Nanoparticle Tracking Analysis, Bioanalyzer, flow cytometry, and LC-MS/MS. The different methods yielded samples of different morphology, particle size, and proteomic profile. For the conditioned medium, Izon 35 isolated the highest number of EV proteins followed by exoEasy, which also isolated fewer non-EV proteins. For the plasma samples, exoEasy isolated a high number of EV proteins and few non-EV proteins, while Izon 70 isolated the most EV proteins. We conclude that no method is perfect for all studies, rather, different methods are suited depending on sample type and interest in EV subtype, in addition to sample volume and budget.

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Section:  Discussionmentioning

confidence: 86%

Molecular evaluation of five different isolation methods for extracellular vesicles reveals different clinical applicability and subcellular origin

Veerman

Teeuwen

Czarnewski

et al. 2021

J of Extracellular Vesicle

197

150

View full text Add to dashboard Cite

show abstract

“…A recent precipitation-based commercial kit demonstrated higher exosome extraction efficiency than ultracentrifugation but with a relatively low purity. 27 Third, PD-L1 may not be influenced by the course of MCC and other exosomal proteins such as HSP70 28,29 or miRNA [30][31][32] may be more relevant.…”

Section: Discussionmentioning

confidence: 99%

“…No gold standard for exosome isolation exists and exosome isolation from serum or plasma is especially problematic because of the small volume available, its high viscosity, and its high concentration of proteins and lipoproteins. A recent precipitation‐based commercial kit demonstrated higher exosome extraction efficiency than ultracentrifugation but with a relatively low purity 27 . Third, PD‐L1 may not be influenced by the course of MCC and other exosomal proteins such as HSP70 28,29 or miRNA 30‐32 may be more relevant.…”

Section: Discussionmentioning

confidence: 99%

PD‐L1 in circulating exosomes of Merkel cell carcinoma

Zanella

Vautrot

Aubin

et al. 2022

Experimental Dermatology

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Exosomes, as potential circulated biomarkers, have recently become a topic of interest in the field of oncology. Immune checkpoint molecule PD‐L1 has recently been detected in circulating exosomes from cancer patients. The purpose of this work was to evaluate PD‐L1 levels in circulating exosomes (Exo‐PD‐L1) isolated from patients’ plasma suffering from Merkel cell carcinoma (MCC). We conducted a prospective bicentric cohort study. PD‐L1 was analysed in circulating exosomes from plasma samples of patients suffering from MCC stage I to IV (according to the AJCC 8). Exosomes from 34 patients corresponding to 66 samples were analysed. PD‐L1 was identified in circulating exosomes of MCC patients. Exo‐PD‐L1 levels of MCC patients were similar to healthy donors and lower than other cancers such as melanoma. Exo‐PD‐L1 levels tended to be higher in MCC patients with distant metastases. Furthermore, Exo‐PD‐L1 levels did not significantly vary over the course of the disease whatever the disease course or the response to treatment. This study assessed the presence of PD‐L1 in circulating exosomes of MCC patients. The low levels of Exo‐PD‐L1 and small changes over the course of the disease may be due to the metastatic dissemination of MCC, which is mainly through the skin and lymph nodes rather than blood. PD‐L1 was identified in circulating exosomes of MCC patients and tends to be higher in advanced disease. This preliminary study is a proof of concept of PD‐L1 detection in circulating exosomes of MCC patients.

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“…The PCR procedure was as follows: Pre-denaturation at 95˚C for 5 min, followed by 40 cycles of denaturation at 95˚C for 15 sec, annealing at 60˚C for 20 sec and extension at 72˚C for 35 sec. GAPDH was used as the internal reference for GATA6 mRNA, and the expression of miR-200a-3p was normalized using cel-miR-39 (25)(26)(27). The data were analyzed based on the 2 -ΔΔCq method (28,29).…”

Section: Reverse Transcription Quantitative Polymerase Chain Reaction...mentioning

confidence: 99%

MicroRNA‑200a‑3p and GATA6 are abnormally expressed in patients with non‑small cell lung cancer and exhibit high clinical diagnostic efficacy

Fang

et al. 2022

Exp Ther Med

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Lung cancer is one of the main threats to human health. Survival of patients with lung cancer depends on timely detection and diagnosis. Among the genetic irregularities that control cancer development and progression, there are microRNAs (miRNAs/miRs). The present study aimed to investigate the expression patterns of miR-200a-3p and transcription factor GATA-6 (GATA6) in peripheral blood of patients with non-small cell lung cancer (NSCLC) and their clinical significance. The expression patterns of miR-200a-3p and GATA6 in the peripheral blood of patients with NSCLC and healthy subjects were measured via reverse transcription-quantitative PCR. The correlation between GATA6/miR-200a-3p expression and their diagnostic efficacy were analyzed by receiver operating characteristic curve analysis. The association between miR-200a-3p/GATA6 expression with the patient clinicopathological characteristics, and their correlation with carcinoembryonic antigen (CEA), neuron specific enolase (NSE) and squamous cell carcinoma antigen (SCCAg) were evaluated. The cumulative survival rate was examined, and whether miR-200a-3p and GATA6 expression levels were independently correlated with the prognosis of NSCLC was analyzed using multivariate logistic regression model. The results demonstrated that the expression of miR-200a-3p was high and that of GATA6 was low in the peripheral blood of patients with NSCLC, and both exhibited high clinical diagnostic efficacy. miR-200a-3p was revealed to target GATA6 by dual-luciferase assay. miR-200a-3p in the peripheral blood was correlated with TNM stage, lymph node metastasis and distal metastasis, while GATA6 in the peripheral blood was correlated with TNM stage and lymph node metastasis. miR-200a-3p and GATA6 were positively correlated with CEA and SCCAg, but not with NSE. High expression of miR-200a-3p and low expression of GATA6 predicted poor prognosis in patients with NSCLC. After adjusting for TNM stage, lymph node metastasis, distance metastasis, GATA6, CEA, NSE and SCCAg in the logistic regression model, it was indicated that the high expression of miR-200a-3p increased the risk of death in patients with NSCLC. Collectively, it was revealed that miR-200a-3p and GATA6 were abnormally expressed in the peripheral blood of patients with NSCLC. Serum levels of miR-200a-3p >1.475 and GATA6 <1.195 may assist the early diagnosis of NSCLC. GATA6 may function in NSCLC as a miR-200a-3p target, which may provide a future reference for NSCLC early diagnosis and treatment.

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Comparison of Serum Exosome Isolation Methods on Co-precipitated Free MicroRNAs

Cited by 5 publications

References 29 publications

Molecular evaluation of five different isolation methods for extracellular vesicles reveals different clinical applicability and subcellular origin

Molecular evaluation of five different isolation methods for extracellular vesicles reveals different clinical applicability and subcellular origin

PD‐L1 in circulating exosomes of Merkel cell carcinoma

MicroRNA‑200a‑3p and GATA6 are abnormally expressed in patients with non‑small cell lung cancer and exhibit high clinical diagnostic efficacy

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