Exosome Isolation

Exosomes and EVs for Liquid Biopsy Biomarker Studies

Exosomes and other extracellular vesicles (EVs) are important mediators of cell-cell communication and they are revolutionizing the field of liquid biopsy as carriers of nucleic acids (DNA, RNA, miRNA, other small RNAs), proteins and lipids that reflect the state of diseases.

The competitive advantage of extracellular vesicles as biomarkers is based mainly on their great stability and integrity and on their high abundance, associated for example with the process of tumor progression.

Tumor-derived extracellular vesicles (EVs) are secreted in large amounts into biological fluids of cancer patients. The analysis of EV´s cargoes has been associated with patient´s outcome and response to therapy. However, current technologies for EV isolation are tedious and inefficient for routine implementation.

Nucleus Biotech is pleased to introduce ExoGAG developed by NEXOTECH (Lugo, Spain), an easy to use and highly efficient new technology to isolate pure EVs.

ExoGAG:  The Next Generation of Exosome Isolation for Biomarker Detection in Liquid Biopsy

Exosome purification for accurate downstream analysis of exosomal proteins and nucleic acids

ExoGAG is a patented extracellular vesicles purification method developed by NEXOTECH (Lugo, Spain) for isolation of EVs from liquid biopsy samples – free of non-EV contaminations.  It enables the accurate analysis of proteins or genetic material contained in the EVs, which is ideal for genuine biomarker detection.

ExoGAG uses a simple precipitation protocol to specifically isolate exosomes and other extracellular vesicles. The ExoGAG technology is based on glycosaminoglycans (GAGs) coating exosomes and other EVs. The negatively charged GAGs are neutralized by the positive charge of cationic agents in the ExoGAG solution leading to a complex that can be easily purified by centrifugation (Herrero et al, Cancers 2019).

• For small liquid biopsy inputs (plasma, serum, urine etc.)

• Easy, fast, and scalable precipitation protocol using a standard lab centrifuge

• Isolates pure exosomes/EVs at high yields

• Ideal for true biomarker detection and screening

After a short incubation with the liquid biopsy sample such as plasma, serum, urine, etc., pure exosomes are isolated by a brief standard centrifugation, whereas non-exosomal protein, DNA, & RNA contaminations are removed. The purified exosomes can be detected by NTA, and can be used to analyze exosome-specific proteins, DNA, mRNA, miRNA and other small RNAs at highest accuracy.

Nanoparticles Tracking Analysis (NTA) of EVs isolated with ExoGAG from 50 ul plasma and Flow Cytometry analysis of exosome-specific CD9 marker in EVs isolated with ExoGAG from 100 ul plasma

Nanoparticles Tracking Analysis (NTA) of EVs isolated with ExoGAG from 50 ul serum and Flow Cytometry analysis of exosome-specific CD63 marker in EVs isolated with ExoGAG from 100 ul serum

Nanoparticles Tracking Analysis (NTA) of EVs isolated with ExoGAG from 3ml urine and Flow Cytometry analysis of exosome-specific CD63 marker in EVs isolated with ExoGAG from 3ml urine

Citations:

Herrero et al, Cancers 2019: Extracellular Vesicles-Based Biomarkers Represent a Promising Liquid Biopsy in Endometrial Cancer

Mercadal et al., Journal of Molecular Sciences 2020: Impact of Extracellular Vesicle Isolation Methods on Downstream miRNA Analysis in Semen: A Comparative Study

Herrero et al, Biomedicines 2023: Extracellular Vesicles’ Genetic Cargo as Noninvasive Biomarkers in Cancer: A Pilot Study Using ExoGAG Technology

ExoGAG-purified Extracellular Vesicles can be used for the following downstream applications:

• NTA

• FACS

• ELISA

• (Glyco-)Protein Microarrays

• qRT-PCR for miRNA/mRNA detection

• BEAMing and Digital PCR for allelic variant and methylation analysis

• DNA-and RNA-seq including Small RNA-seq

Exemplary Data:

miRNA normalized Cq RT-qPCR values using RNA from EVs isolated from 500 ul plasma and 500 ul saliva by ExoGAG, and RNA isolated from Total Saliva

mRNA qRT-PCR Cq values with RNA obtained from EVs isolated from 500 ul or 3ml of plasma and 3ml of urine

Mutant Allele Fraction (%) determined by ddPCR with cfDNA from 5ml Plasma or with evDNA extracted from EVs isolated from 500 ul plasma by ExoGAG

Methylation analysis by ddPCR of genomic DNA of different CRC cell lines and their EVs isolated from 2ml of culture medium using ExoGAG