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Extracellular
vesicles (EVs) are tiny
membrane-bound particles released by
cells that carry proteins, lipids, and
genetic material. Because EVs protect
their contents from degradation and
reflect the biological state of the
cells that produced them, they have
attracted considerable interest as a
source of biomarkers for liquid
biopsies. Among the molecules carried by
EVs, microRNAs (miRNAs) are especially
promising because their expression
patterns can reveal important
information about disease processes.
Researchers at the
Technical University of Munich summarize
the current landscape of EV-associated
miRNA analysis and discuss the
challenges that must be overcome before
these biomarkers can be routinely used
in clinical diagnostics.
EV-associated
miRNAs can be detected in
many body fluids, including blood,
urine, saliva, breast milk, respiratory
samples, cerebrospinal fluid and many
more ... These readily accessible
samples offer a minimally invasive
alternative to tissue biopsies and may
provide valuable insights into cancer,
neurological disorders, and other
diseases.
The researchers explain
how EVs are formed and how specific
miRNAs are packaged into these vesicles.
Understanding these biological processes
is important because the composition of
EV cargo influences their diagnostic
value. The review also highlights the
wide variety of methods available for EV
isolation and characterization, along
with approaches used to extract and
quantify EV-associated RNA.
Modern analytical
technologies such as RNA sequencing and
RT-qPCR have greatly improved
researchers’ ability to measure miRNAs
from very small samples. However,
differences in sample collection, EV
isolation procedures, and data analysis
methods can introduce variability that
complicates comparisons between studies.
The researchers emphasize
the importance of standardization
efforts, including the MISEV guidelines,
MIQE
recommendations, EV-TRACK,
and EV Task Force biofluid guidelines.
These initiatives aim to improve
reproducibility and establish best
practices for sample handling, quality
assessment, and data normalization.
Another challenge involves
the complexity of biological samples.
Variations between patients, low RNA
abundance, and differences in miRNA
isoforms require sophisticated
computational approaches and
multivariate analysis methods. The
researchers discuss emerging strategies
to address these issues and improve the
reliability of biomarker discovery.
Ultimately, the
researchers propose an integrated
workflow that spans every stage of the
process, from liquid biopsy collection
to the identification of clinically
actionable EV-miRNA signatures.
Establishing standardized procedures and
robust analytical methods will be
essential for translating EV-based
diagnostics from research laboratories
into routine clinical practice.
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Extracellular
vesicle (EV)-associated microRNAs
(miRNAs) are promising minimally
invasive biomarkers, as EV encapsulation
protects miRNAs from degradation while
preserving disease- and cell-
type-specific expression patterns across
diverse human body fluids. This review
summarizes the current understanding of
miRNA and EV biogenesis, including the
mechanisms governing EV cargo packaging.
We further discuss the broad range of EV
sources used in liquid biopsy and
molecular diagnostics, including blood,
urine, saliva, milk, respiratory fluids,
and cerebrospinal fluid, highlighting
both the diagnostic utility and key
pre-analytical challenges associated
with EV-miRNA analysis. State-of-the-art
EV isolation approaches, including
differential ultracentrifugation,
size-exclusion chromatography,
affinity-based methods, microfluidic
platforms, flow cytometry and
nanoparticle-based analyses are not
isolation methods and are therefore
excluded from this comparison. In
addition, workflows for EV-associated
RNA extraction and quantification,
including RNA sequencing and reverse
transcription-quantitative polymerase
chain reaction-based methodologies, are
critically evaluated. Emphasis is placed
on challenges related to data quality
assessment, normalization strategies,
low-input sample analysis, and high
sample heterogeneity. This review also
summarizes current standardization
initiatives, including MISEV, MIQE,
EV-TRACK, and EV Task Force biofluid
guidelines, emphasizing the importance
of rigorous reporting standards,
harmonized pre-analytical workflows, and
multiparametric normalization strategies
for reproducible detection and accurate
quantification. Finally, we discuss
emerging best practices and unresolved
challenges in multivariate modeling,
miRNA isoform-based analyses, and in
silico validation of the target
recognition elements and regulatory
pathways. An integrated, guideline-based
workflow from liquid biopsy collection
to clinically actionable EV-miRNA
signatures is proposed to facilitate
translation of this approach into
routine molecular diagnostics.
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Diagnostic and
Therapeutic Roles of Extracellular
Vesicles in Chronic Kidney Disease: A
Systematic Review
Tunahan Ergunay, Alessia Brossa, Benedetta
Bussolati
J Extracell Vesicles 2026 15(5): e70300
Extracellular
vesicles (EVs) are increasingly
investigated across a wide range of
diseases as potential biomarkers and
therapeutic tools. To date, EVs have
been isolated from diverse sources,
including urine, blood, saliva, tissue,
and cell cultures, with research
focusing primarily on their protein and
RNA cargo. Owing to their non-invasive
accessibility, selective cargo loading,
and molecular richness, biological
fluid-derived EVs have been proposed as
promising candidates for biomarker
discovery. In addition, several studies
have explored EVs for therapeutic
purposes, either by direct
administration to diseased cells or
organisms, by engineering them to
enhance their efficacy, or by targeting
them to modulate pathological processes.
In this systematic review, we synthesize
current evidence on the diagnostic and
therapeutic roles of EVs in CKD and
related conditions, integrating findings
across different EV sources, cargos, and
disease models, and providing an
integrated perspective on the role of
EVs in chronic kidney disease-related
research. By comparing molecular
findings from diagnostic and therapeutic
studies, we also identify key
overlapping pathways and biological
processes that may represent relevant
mechanistic frameworks. Recognizing
these convergent pathways can help unify
data and guide future research toward
mechanism-driven, and clinically
translatable EV applications in
nephrology.
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Enhanced Early
Detection of Colorectal Cancer via
Blood Biomarker Combinations
Identified Through Extracellular
Vesicle Isolation and Artificial
Intelligence Analysis
Bonhan Koo, Young Il Kim, Minju Lee,
Seok-Byung Lim, Yong Shin
J Extracell Vesicles 2025 14(6): e70088
Colorectal
cancer (CRC) remains a major cause of
cancer-related deaths worldwide, with
early detection being crucial for
improving survival rates. Despite the
potential of extracellular vesicles
(EVs) as blood biomarkers for CRC
diagnosis, their clinical utility is
limited due to complex and
time-consuming isolation methods,
unverified biomarkers and low diagnostic
performance. Here, we introduce the
ZAHV-AI system, which combines the
zeolite-amine and homobifunctional
hydrazide-based extracellular vesicle
isolation (ZAHVIS) platform with
AI-driven analysis for enhanced CRC
diagnosis. The ZAHVIS platform enables
simple, rapid and cost-effective EV
isolation and one-step extraction of
EV-derived proteins and nucleic acids
(NAs), providing a streamlined approach.
Using blood plasma samples from 80 CRC
patients across all stages and 20
healthy individuals, we identified four
EV-derived miRNA blood biomarkers
(miR-23a-3p, miR-92a-3p, miR-125a-3p and
miR-150-5p) by confirming statistical
significance with relative
quantification (RQ) values from
real-time PCR and integrated these with
carcinoembryonic antigen (CEA) levels
into an AI-driven diagnostic model.
Among 31 combinations used to identify
optimal sets, optimal combination
(miR-23a-3p, miR-92a-3p, miR-150-5p and
CEA) for overall CRC achieved an area
under the curve (AUC) of 0.9861,
outperforming individual markers and
conventional CEA tests. Notably, the
system achieved perfect performance in
detecting stages 0-1 (AUC: 1.0) and
demonstrated high accuracy for stage 2
(AUC: 0.9722) and early-stage CRC (AUC:
0.9861), using stage-specific optimal
combinations. Therefore, the ZAHV-AI
system offers a reliable and clinically
relevant tool for CRC diagnostics,
significantly enhancing early detection
and monitoring capabilities.
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Toward
reliable biomarker signatures in the
age of liquid biopsies - how to
standardize the small RNA-Seq workflow
Buschmann D, Haberberger A, Kirchner B,
Spornraft M, Riedmaier I, Schelling G,
Pfaffl MW
Nucleic Acids Res. 2016 44(13): 5995-6018
Small
RNA-Seq has emerged as a powerful tool
in transcriptomics, gene expression
profiling and biomarker discovery.
Sequencing cell-free nucleic acids,
particularly microRNA (miRNA), from
liquid biopsies additionally provides
exciting possibilities for molecular
diagnostics, and might help establish
disease-specific biomarker signatures.
The complexity of the small RNA-Seq
workflow, however, bears challenges and
biases that researchers need to be aware
of in order to generate high-quality
data. Rigorous standardization and
extensive validation are required to
guarantee reliability, reproducibility
and comparability of research findings.
Hypotheses based on flawed experimental
conditions can be inconsistent and even
misleading. Comparable to the
well-established MIQE guidelines for
qPCR experiments, this work aims at
establishing guidelines for experimental
design and pre-analytical sample
processing, standardization of library
preparation and sequencing reactions, as
well as facilitating data analysis. We
highlight bottlenecks in small RNA-Seq
experiments, point out the importance of
stringent quality control and
validation, and provide a primer for
differential expression analysis and
biomarker discovery. Following our
recommendations will encourage better
sequencing practice, increase
experimental transparency and lead to
more reproducible small RNA-Seq results.
This will ultimately enhance the
validity of biomarker signatures, and
allow reliable and robust clinical
predictions.
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How Laboratory
Guidelines Promote the Validity of
Circulating Extracellular
Vesicle-Associated Nucleic Acid
Biomarker Signatures in Liquid Biopsy
Michael W Pfaffl
Int J Mol Sci 2025 26(24): 12115
Circulating nucleic acids, particularly
those associated with extracellular
vesicles (EVs), represent a promising
class of molecular biomarkers in liquid
biopsy for 'non-invasive' disease
diagnostics, for better prognosis, and for
therapeutic monitoring. However, the
translation of this new circulating
biomarker source into clinical practice is
mostly hindered by methodological
variability and a lack of standardization
across the analytical workflow. This
article highlights the implementation of
international academic guidelines, such as
Minimum Information for Publication of
Quantitative Real-Time PCR Experiments
(MIQE) and Minimal Information for Studies
of Extracellular Vesicles (MISEV), in the
entire analytical procedure in promoting
the integrity, reproducibility, and
validity of EV-associated nucleic acid
markers in molecular diagnostics. By
standardizing the liquid biopsy workflow
from tissue sampling up to data analysis
and statistics, these established
guidelines lay the necessary scientific
basis for a robust, reproducible,
reliable, and valid RNA and DNA biomarker
discovery in EVs. The ultimate goal is the
successful implementation of the developed
biomarker signature into the clinical
diagnostic routine, but this requires
further rounds of rigorous validation. The
regularly updated guidelines should not be
seen as optional recommendations, but more
like an essential pillars of scientific
rigor and standardization in order to
achieve better and biological meaningful
biomarker results in liquid biopsy.
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SECmeres outperform
extracellular vesicles as potential
blood RNA biomarkers for Alzheimer's
disease
Edgar Gonzalez-Kozlova, Swapnil Tichkule,
Yohei Nose, Tzu-Yi Chen, Eduard Reznik,
Juliet V Santiago Anish Korrapati, Taliah
Soleymani, Roman Kosoy, Igor Figueiredo,
Donghoon Lee, Gabriel E Hoffman, Natasha
Kyprianou, Ronald E Gordon, Carlos
Cordon-Cardo, Srikant Rangaraju, Nicholas
T Seyfried, Vahram Haroutunian, John F
Fullard, Panos Roussos, Navneet Dogra
Nature Communications 2026 17(1): 5453
Cells
release heterogeneous extracellular
vesicles and particles (EVPs) into
circulation, carrying RNA and proteins
that reflect their origin. Recently,
brain-derived EVs have gained
significant attention as non-invasive
biomarkers for Alzheimer's disease (AD).
Here, we identified sub-50nm
extracellular nanoparticles in human
brain and blood that lack the hallmarks
of small EVs, exosomes, exomeres, and
supermeres but are enriched for
brain-specific markers, hereafter termed
small EPs or 'SECmeres'. We discovered
that RNAs associated with SECmeres
discriminated AD cases from controls
with higher significance than small EVs,
large EVs showed no differences.
Discriminating RNAs were enriched in
small EVs (Synaptotagmin,
Alpha-synuclein, MAPT) or SECmeres
(L1CAM, Syntaxin, Neurogranin),
indicating distinct brain-derived
signatures. Single-cell RNAseq
deconvolution shows small EVs contain
RNAs from diverse brain cells, whereas
SECmeres enrich brain endothelial
transcripts, lining cerebral blood
vessels and forming the blood-brain
barrier (BBB). These findings challenge
the prevailing view that small EVs are
the primary carriers of biomarkers.
Collectively, our study shows that blood
EVPs carry brain-specific information
for liquid biopsy, pending validation in
larger blinded clinical trials.
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Defining the
Parameters for Sorting of RNA Cargo
Into Extracellular Vesicles
Ahmed Abdelgawad, Yiyao Huang, Olesia
Gololobova, Yanbao Yu, Kenneth W Witwer,
Vijay Parashar, Mona Batish
J Extracell Vesicles 2025 14(7): e70113
Extracellular
vesicles (EVs) are small particles that
are released by cells and mediate
cell-cell communication by transferring
bioactive molecules such as RNA. RNA
cargo of EVs, including coding and
non-coding RNAs, can change the
behaviour of recipient cells, affecting
processes including gene expression,
proliferation, and Fapoptosis. CircRNAs
are stable and resistant to degradation
and have been shown to be enriched in
EVs. They play key roles in gene
regulation and are also emerging as
promising biomarkers for disease
diagnosis due to their stability and
disease-specific expression. Although
microRNAs (miRNAs) are the most well
studied RNA cargo of EVs, very little is
known about the mechanisms of enrichment
of circular RNAs (circRNAs) as well as
long linear RNAs. Here, we take a
comprehensive genome-wide approach to
investigate the role of structuredness
and shape along with GC%, size, exon
count and coding potential, in the
sorting and enrichment of circular and
long linear RNAs into EVs. We developed
a model using these parameters to
predict the likelihood of EV packaging
of RNA and it was validated by using
single molecule RNA imaging of EV bound
RNAs. Furthermore, we found that
structuredness could explain the
relative enrichment of circRNAs over
their linear counterparts. These results
were validated on existing public
databases of circular and linear RNAs in
EVs. By identifying and analysing these
factors, we aim to better understand the
complex mechanisms behind EV-mediated
RNA transfer and its impact on cell
communication in both health and
disease. This mechanistic understanding
of RNA enrichment in EVs is crucial for
engineering EVs with selective RNA
cargo.
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MISEV and MIQE:
integrating domain-specific and
general standards to strengthen
extracellular vesicle biomarker
research
Michael W Pfaffl, Mikael Kubista, Jo
Vandesompele, Stephen A Bustin
Extracell Vesicles Circ Nucl Acids 2025
6(4): 669-676
Extracellular vesicles (EVs) have
significant potential as therapeutic
agents and as sources of diagnostic,
predictive, and prognostic nucleic acid
biomarkers. However, variability in EV
workflows and inadequate standardization
of downstream analysis pose major
obstacles to reproducibility. The MISEV
(Minimal Information for Studies of
Extracellular Vesicles) guidelines provide
essential domain-specific recommendations
for EV isolation, characterization,
analysis, nomenclature, and reporting, but
deliberately refrain from prescribing
methods for the molecular quantification
of EV cargo. Among the analytical
platforms used in EV studies, quantitative
reverse transcription PCR (RT-qPCR) is the
most critical method for validating and
quantifying EV-associated RNA. The
recently revised MIQE (Minimum Information
for Publication of Quantitative Real-Time
PCR Experiments) 2.0 guidelines offer a
detailed foundation for ensuring
analytical validity in RT-qPCR-based
quantitative applications. The proposed
model of harmonizing general and
domain-specific guidelines provides a
scalable blueprint for improving
reproducibility across complex biomarker
development workflows in molecular
diagnostics.
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Obstacles
and opportunities in the
functional analysis of
extracellular vesicle RNA - an
ISEV position paper
Mateescu
B, Kowal EJ, van Balkom BW, Bartel S,
Bhattacharyya SN, Buzás EI, Buck AH, de
Candia P, Chow FW, Das S, Driedonks TA,
Fernández-Messina L, Haderk F, Hill AF,
Jones JC, Van Keuren-Jensen KR, Lai CP,
Lässer C, Liegro ID, Lunavat TR,
Lorenowicz MJ, Maas SL, Mäger I,
Mittelbrunn M, Momma S, Mukherjee K,
Nawaz M, Pegtel DM, Pfaffl MW,
Schiffelers RM, Tahara H, Théry C, Tosar
JP, Wauben MH, Witwer KW, Nolte-'t Hoen
EN
J Extracell Vesicles. 2017 6(1):
1286095 -- eCollection 2017
The
release of RNA-containing extracellular
vesicles (EV) into the extracellular
milieu has been demonstrated in a
multitude of different in vitro cell
systems and in a variety of body fluids.
RNA-containing EV are in the limelight
for their capacity to communicate
genetically encoded messages to other
cells, their suitability as candidate
biomarkers for diseases, and their use
as therapeutic agents. Although EV-RNA
has attracted enormous interest from
basic researchers, clinicians, and
industry, we currently have limited
knowledge on which mechanisms drive and
regulate RNA incorporation into EV and
on how RNA-encoded messages affect
signalling processes in EV-targeted
cells. Moreover, EV-RNA research faces
various technical challenges, such as
standardisation of EV isolation methods,
optimisation of methodologies to isolate
and characterise minute quantities of
RNA found in EV, and development of
approaches to demonstrate functional
transfer of EV-RNA in vivo. These topics
were discussed at the 2015 EV-RNA
workshop of the International Society
for Extracellular Vesicles. This
position paper was written by the
participants of the workshop not only to
give an overview of the current state of
knowledge in the field, but also to
clarify that our incomplete knowledge -
of the nature of EV(-RNA)s and of how to
effectively and reliably study them -
currently prohibits the implementation
of gold standards in EV-RNA research. In
addition, this paper creates awareness
of possibilities and limitations of
currently used strategies to investigate
EV-RNA and calls for caution in
interpretation of the obtained data.
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Extracellular
MicroRNAs: A Stable and Diverse Source
of Transcriptional Control
Megan I Mitchell and Olivier Loudig
Biomolecules 2026 16(6): 787
MicroRNAs
(miRNAs) are a highly conserved class of
small (19-25 nucleotides) non-coding
RNAs that play critical roles in
post-translational gene regulation.
Dysregulation of miRNA expression has
been widely implicated in the
development and progression of numerous
diseases, particularly cancer,
positioning them as promising candidates
for diagnostic and prognostic
applications. In parallel, miRNAs are
frequently detected in extracellular
vesicles (EVs), where they contribute to
intercellular communication and have
emerged as attractive non-invasive
biomarkers. Importantly, EV-associated
miRNA profiles do not always directly
mirror intracellular miRNA abundance.
While altered cellular expression can
influence EV-miRNA content, selective
and regulated sorting mechanisms also
actively shape EV cargo composition.
These include sequence- and motif-based
recognition elements (such as
EXOmotifs), RNA-binding proteins
(including hnRNPA2B1, YBX1, and
SYNCRIP), and lipid-associated pathways
such as ceramide-dependent mechanisms.
Together, these processes enable the
preferential packaging of specific
miRNAs into EVs, independent of their
relative cellular expression levels.
This review therefore integrates both
perspectives: it summarizes current
evidence supporting dysregulated miRNAs
detected in EVs as disease-associated
biomarkers and critically examines the
molecular mechanisms governing miRNA
sorting into EVs. By clarifying the
interplay between cellular miRNA
dysregulation and active EV loading
processes, we highlight the complexity
underlying EV-miRNA signatures and
underscore the need for standardized
mechanistic frameworks to improve their
translational utility in cancer
diagnostics and beyond.
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A pipeline for the
development and analysis of
extracellular vesicle-based
transcriptomic biomarkers in molecular
diagnostics
Christian Grätz, Martina Schuster, Florian
Brandes, Agnes S Meidert, Benedikt
Kirchner, Marlene Reithmair, Gustav
Schelling, Michael W Pfaffl
Mol Aspects Med 2024 (97): 101269
Extracellular vesicles are shed by every
cell type and can be found in any
biofluid. They contain different
molecules that can be utilized as
biomarkers, including several RNA
species which they protect from
degradation. Here, we present a pipeline
for the development and analysis of
extracellular vesicle-associated
transcriptomic biomarkers that our group
has successfully applied multiple times.
We highlight the key steps of the
pipeline and give particular emphasis to
the necessary quality control
checkpoints, which are linked to
numerous available guidelines that
should be considered along the workflow.
Our pipeline starts with patient
recruitment and continues with blood
sampling and processing. The
purification and characterization of
extracellular vesicles is explained in
detail, as well as the isolation and
quality control of extracellular
vesicle-associated RNA. We point out the
possible pitfalls during library
preparation and RNA sequencing and
present multiple bioinformatic tools to
pinpoint biomarker signature candidates
from the sequencing data. Finally,
considerations and pitfalls during the
validation of the biomarker signature
using RT-qPCR will be elaborated.
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Summary of the ISEV workshop on
extracellular vesicles as disease
biomarkers
A
ISEV meeting Report -- held in
Birmingham, UK, during December 2017
Aled
Clayton, Dominik Buschmann, J. Brian
Byrd, David R. F. Carter, Lesley Cheng,
Carolyn Compton, George Daaboul, Andrew
Devitt, Juan Manuel Falcon-Perez, Chris
Gardiner, Dakota Gustafson, Paul
Harrison, Clemens Helmbrecht, An
Hendrix, Andrew Hill, Andrew Hoffman,
Jennifer C. Jones, Raghu Kalluri, Ji
Yoon Kang, Benedikt Kirchner, Cecilia
Lässer, Charlotte Lawson, Metka Lenassi,
Carina Levin, Alicia Llorente, Elena S.
Martens-Uzunova, Andreas Möller, Luca
Musante, Takahiro Ochiya, Ryan C Pink,
Hidetoshi Tahara, Marca H. M. Wauben,
Jason P. Webber, Joshua A. Welsh,
Kenneth W. Witwer, Hang Yin & Rienk
Nieuwland
Journal of Extracellular Vesicles
2018 (7)1
This
report summarises the presentations and
activities of the ISEV Workshop on
extracellular vesicle biomarkers held in
Birmingham, UK during December 2017.
Among the key messages was broad
agreement about the importance of
biospecimen science. Much greater
attention needs to be paid towards the
provenance of collected samples. The
workshop also highlighted clear gaps in
our knowledge about pre-analytical
factors that alter extracellular
vesicles (EVs). The future utility of
certified standards for credentialing of
instruments and software, to analyse EV
and for tracking the influence of
isolation steps on the structure and
content of EVs were also discussed.
Several example studies were presented,
demonstrating the potential utility for
EVs in disease diagnosis, prognosis,
longitudinal serial testing and
stratification of patients. The
conclusion of the workshop was that more
effort focused on pre-analytical issues
and benchmarking of isolation methods is
needed to strengthen collaborations and
advance more effective biomarkers.
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Circulating lncRNA as
Biomarkers and Therapeutics in Oral
Cancer: Silent Scripts with Loud
Impact
Riddhiman Bhattacharyya, Chandra Biswas,
Md Sadi Khan, Sankhadeep Dutta, Subhayan
Sur
Mol Biol Rep 2026 53(1): 783
Oral
squamous cell carcinoma (OSCC) remains a
major global health challenge due to
late diagnosis, high recurrence, and
limited therapeutic success. This review
focuses on circulating long non-coding
RNAs (lncRNAs) as emerging biomarkers
and therapeutic targets that can
transform the clinical management of
OSCC. Unlike earlier studies restricted
to tissue-based analyses, this work
emphasizes circulating lncRNAs
detectable in saliva, plasma, and serum,
highlighting their non-invasive
diagnostic potential and mechanistic
relevance in tumor progression,
metastasis, and therapy resistance.
Relevant studies published up to 2025
were systematically reviewed from PubMed
and Google Scholar to synthesize current
knowledge on their biogenesis, secretion
through extracellular vesicles and
RNA-binding proteins, detection
technologies, and clinical
applicability. Several circulating
lncRNAs, including HOTAIR, MALAT1, MEG3,
ANRIL, and LINC00657, show strong
associations with tumor stage,
prognosis, and therapeutic outcomes. The
review also discusses emerging RNA-based
therapeutic approaches such as antisense
oligonucleotides, RNA interference, and
CRISPR-based gene editing. Overall, the
analysis underscores the diagnostic
precision, prognostic power, and
therapeutic promise of circulating
lncRNAs, proposing them as key
components in the future of liquid
biopsy-driven, personalized oncology for
OSCC.
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Exhaled breath
condensate contains EVs that carry
miRNA cargos of lung tissue origin
that can be selectively purified and
analyzed
Megan I Mitchell, Iddo Z Ben-Dov, Kenny
Ye, Christina Liu, Miao Shi, Ali Sadoughi,
Chirag Shah, Taha Siddiqui, Aham Okorozo,
Martin Gutierrez, Rashmi Unawane, Lisa
Biamonte, Kaushal Parikh, Simon Spivack,
Olivier Loudig
J Extracell Vesicles 2024 13(4): e12440
Lung
diseases, including lung cancer, are
rising causes of global mortality.
Despite novel imaging technologies and
the development of biomarker assays, the
detection of lung cancer remains a
significant challenge. However, the lung
communicates directly with the external
environment and releases aerosolized
droplets during normal tidal
respiration, which can be collected,
stored and analzsed as exhaled breath
condensate (EBC). A few studies have
suggested that EBC contains
extracellular vesicles (EVs) whose
microRNA (miRNA) cargos may be useful
for evaluating different lung
conditions, but the cellular origin of
these EVs remains unknown. In this
study, we used nanoparticle tracking,
transmission electron microscopy,
Western blot analyses and super
resolution nanoimaging (ONi) to detect
and validate the identity of exhaled EVs
(exh-EVs). Using our customizable
antibody-purification assay, EV-CATCHER,
we initially determined that exh-EVs can
be selectively enriched from EBC using
antibodies against three tetraspanins
(CD9, CD63 and CD81). Using ONi we also
revealed that some exh-EVs harbour
lung-specific proteins expressed in
bronchiolar Clara cells (Clara Cell
Secretory Protein [CCSP]) and Alveolar
Type II cells (Surfactant protein C
[SFTPC]). When conducting miRNA next
generation sequencing (NGS) of airway
samples collected at five different
anatomic levels (i.e., mouth rinse,
mouth wash, bronchial brush,
bronchoalveolar lavage [BAL] and EBC)
from 18 subjects, we determined that
miRNA profiles of exh-EVs clustered
closely to those of BAL EVs but not to
those of other airway samples. When
comparing the miRNA profiles of EVs
purified from matched BAL and EBC
samples with our three tetraspanins
EV-CATCHER assay, we captured
significant miRNA expression differences
associated with smoking, asthma and lung
tumor status of our subjects, which were
also reproducibly detected in EVs
selectively purified with our
anti-CCSP/SFTPC EV-CATCHER assay from
the same samples, but that confirmed
their lung tissue origin. Our findings
underscore that enriching exh-EV
subpopulations from EBC allows
non-invasive sampling of EVs produced by
lung tissues.
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Evaluation
of serum extracellular vesicle
isolation methods for profiling miRNAs
by next-generation sequencing
Buschmann
D, Kirchner B, Hermann S, Märte M,
Wurmser C, Brandes F, Kotschote S, Bonin
M, Steinlein OK, Pfaffl MW, Schelling G,
Reithmair M
J Extracell Vesicles. 2018 7(1):
1481321 -- eCollection 2018
Extracellular
vesicles
(EVs) are intercellular communicators
with key functions in physiological and
pathological processes and have recently
garnered interest because of their
diagnostic and therapeutic potential.
The past decade has brought about the
development and commercialization of a
wide array of methods to isolate EVs
from serum. Which subpopulations of EVs
are captured strongly depends on the
isolation method, which in turn
determines how suitable resulting
samples are for various downstream
applications. To help clinicians and
scientists choose the most appropriate
approach for their experiments,
isolation methods need to be
comparatively characterized. Few
attempts have been made to
comprehensively analyse vesicular
microRNAs (miRNAs) in patient biofluids
for biomarker studies. To address this
discrepancy, we set out to benchmark the
performance of several isolation
principles for serum EVs in healthy
individuals and critically ill patients.
Here, we compared five different methods
of EV isolation in combination with two
RNA extraction methods regarding their
suitability for biomarker
discovery-focused miRNA sequencing as
well as biological characteristics of
captured vesicles. Our findings reveal
striking method-specific differences in
both the properties of isolated vesicles
and the ability of associated miRNAs to
serve in biomarker research. While
isolation by precipitation and membrane
affinity was highly suitable for
miRNA-based biomarker discovery, methods
based on size-exclusion chromatography
failed to separate patients from healthy
volunteers. Isolated vesicles differed
in size, quantity, purity and
composition, indicating that each method
captured distinctive populations of EVs
as well as additional contaminants. Even
though the focus of this work was on
transcriptomic profiling of EV-miRNAs,
our insights also apply to additional
areas of research. We provide guidance
for navigating the multitude of EV
isolation methods available today and
help researchers and clinicians make an
informed choice about which strategy to
use for experiments involving critically
ill patients.
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http://evtrack.org
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EV-TRACK
-- transparent reporting
and centralizing knowledge in
extracellular vesicle
research.
EV-TRACK Consortium, Van Deun J,
Mestdagh P, Agostinis P, ... and
much more, Vandesompele J, Hendrix
A; Nat Methods. 2017 14(3):
228-232
We
argue that the field of
extracellular vesicle (EV)
biology needs more transparent
reporting to facilitate
interpretation and replication
of experiments. To achieve this,
we describe EV-TRACK, a
crowdsourcing knowledgebase (http://evtrack.org)
that centralizes EV biology and
methodology with the goal of
stimulating authors, reviewers,
editors and funders to put
experimental guidelines into
practice.
Is
your article EV-TRACKed?
Van Deun J, Hendrix A, and the
EV-TRACK consortium
J Extracell Vesicles. 2017 Nov
10;6(1): 1379835
The EV-TRACK knowledgebase is
developed to cope with the need
for transparency and rigour to
increase reproducibility and
facilitate standardization of
extracellular vesicle (EV)
research. The knowledgebase
includes a checklist for authors
and editors intended to improve
the transparency of
methodological aspects of EV
experiments, allows queries and
meta-analysis of EV experiments
and keeps track of the current
state of the art. Widespread
implementation by the EV
research community is key to its
success.
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