A Comprehensive Study of Vesicular and Non-Vesicular miRNAs from a Volume of Cerebrospinal Fluid Compatible with Clinical Practice

Abstract

Cerebrospinal fluid (CSF) microRNAs (miRNAs) have emerged as potential biomarkers for minimally invasive diagnosis of central nervous system malignancies. However, despite significant advances in recent years, this field still suffers from poor data reproducibility. This is especially true in cases of infants, considered a new subject group. Implementing efficient methods to study miRNAs from clinically realistic CSF volumes is necessary for the identification of new biomarkers. Methods : We compared six protocols for characterizing miRNAs, using 200-µL CSF from infants (aged 0-7). Four of the methods employed extracellular vesicle (EV) enrichment step and the other two obtained the miRNAs directly from cleared CSF. The efficiency of each method was assessed using real-time PCR and small RNA sequencing. We also determined the distribution of miRNAs among different CSF shuttles, using size-exclusion chromatography. Results : We identified 281 CSF miRNAs from infants. We demonstrated that the miRNAs could be efficiently detected using only 200 µL of biofluid in case of at least two of the six methods. In the exosomal fraction, we found 12 miRNAs that might be involved in neurodevelopment. Conclusion : The Norgen and Invitrogen protocols appear suitable for the analysis of a large number of miRNAs using small CSF samples.

Nanoparticles in 472 Human Cerebrospinal Fluid: Changes in Extracellular Vesicle Concentration and miR-21 Expression as a Biomarker for Leptomeningeal metastasis

“…It remains technical problem of evaluating CSF microRNA expression level due to both the absence of endogenous control for normalization and the origin of microRNA (exosomal vs. non-exosomal) [11,12]. Recently, Parieto-Fernandez et al performed a comprehensive measurement of CSF microRNA differentiating vesicular from non-vesicular compartment [36]. According to their study, miR-21 existed mainly in non-vesicular (CD63 negative) fraction and was nearly abolished with proteinase and RNase combined treatment.…”

“…However, we could not explain why the miR-21 expression pattern became signi cantly different between EV-decreased and EV-increased groups not by RNA amount but by NTA-measure EV concentration at this study design. Very low concentration of CSF extracellular RNA may give inconsistent results according to different extraction and RT-PCR methods [36,37]. We expect more sophisticated EV extraction method keeping non-vesicular proteins and microRNAs from undesirable loss or damage and solid standard RNA extraction and RT-PCR process could help to solve this question in future.…”

Nanoparticles in 472 Human Cerebrospinal Fluid: Changes in Extracellular Vesicle Concentration and miR-21 Expression as a Biomarker for Leptomeningeal Metastasis

“…It remains a technical problem of evaluating CSF microRNA expression level due to both the absence of endogenous control for normalization and the origin of microRNA (exosomal vs. non-exosomal) [ 11 , 12 ]. Recently, Parieto-Fernandez et al performed a comprehensive measurement of CSF microRNA differentiating vesicular from non-vesicular compartment [ 36 ]. According to their study, miR-21 existed mainly in non-vesicular (CD63 negative) fraction and was nearly abolished with proteinase and RNase combined treatment.…”

“…However, we could not explain why the miR-21 expression pattern became significantly different between the EV-decreased and EV-increased groups not by RNA amount but by NTA-measure EV concentration at this study design. A very low concentration of CSF extracellular RNA may give inconsistent results according to different extraction and RT-PCR methods [ 36 , 37 ]. We expect a more sophisticated EV extraction method keeping non-vesicular proteins and microRNAs from undesirable loss or damage, and solid standard RNA extraction and RT-PCR process could help to solve this question in future.…”

Extracellular Vesicle miRNAs in the Promotion of Cardiac Neovascularisation

“…An argument against the presence of Ago2 in EVs is that serum, a typical cell culture additive, contains a significant amount of non-vesicular Ago2 which may contaminate crude EV pellets prepared by standard EV isolation techniques, including ultracentrifugation of the conditioned medium at 100,000 × g (Huang et al, 2013 ). Size exclusion chromatography or density gradient can be used to separate vesicular from non-vesicular components of cell culture media (Prieto-Fernández et al, 2019 ). Jeppesen et al ( 2019 ) recently used high-resolution density gradients to separate a crude small EV pellet into non-vesicular and vesicular fractions.…”