Rich in micro-RNA and other short-fragmented RNA sequences essential for cell-to-cell communication, exosomes have emerged as pivotal components in pathology and disease progression.
Image Credit: Norgen Biotek Corp.
Despite the challenges associated with studying exosomes due to their low abundance, small size, and fragility, they are present throughout the human body and can be purified from various sample types, including plasma, serum, urine, amniotic fluid, cerebrospinal fluid (CSF) and saliva.
This article addresses the three common challenges related to exosome purification and offers recommendations and solutions for these issues.
Low yield
Exosomes are present in low concentrations within liquid biopsy samples, making their purification challenging.
Utilizing proprietary isolation technology, Norgen can achieve high yields of exosomes from samples for downstream applications, such as Western blotting, Nanosight (NTA), Electron Microscopy, and RNA purification, all while preserving the integrity and contents of the exosomes.
This is supported by a 2020 study by Reale et al., which demonstrated that Norgen’s exosome purification kit yielded superior quantities of extracellular vesicles (EVs). The study further confirmed the identity of these EVs as exosomes by correlating this finding with levels of the exosome-specific protein TSG101.
Additionally, Alex Rolland, co-founder and chief scientist at Cancer Treatment Options and Management Inc. (CTOAM), noted the improvement in isolation quantity after switching to Norgen’s product:
“Recently, Norgen has worked with us to purify blood-based exosomes and exosomal-RNA RNA at a high enough concentration to allow RNA sequence-based expression testing… we are able to obtain expression of 20,000 genes from tumor-associated exosomes from cancer patients both in remission and experiencing recurrence.”
Contamination
Exosomes often co-purify with other extracellular vesicles and similar structures due to their comparable sizes, which can compromise exosome purity and affect downstream applications as well as the reliability of results.
Many widely used exosome purification methods, such as ultracentrifugation, precipitation and filtration, are size-based and consequently susceptible to such contamination.
Specifically, the industry-standard ultracentrifugation frequently results in a single pellet containing a mixture of small vesicles, total RNA and small RNA, leading to contaminated outcomes. Similar contamination issues also arise with polyethylene glycol (PEG) precipitation, a method commonly employed in many commercial kits.
Norgen’s Exosome Purification Kits circumvent ultracentrifugation and PEG precipitation by leveraging its proprietary isolation technology that targets the charge of exosomal surface proteins.
One of the most effective methods for exosome purification is antibody purification, which specifically targets exosome surface proteins.
A recent comparison of microRNA diversity from size-based separation (Ultracentrifugation, ExoQuick, and exoEasy) versus surface protein-based isolation methods (Miltenyi Antibodies and Norgen) demonstrated that Norgen’s proprietary SiC resin matrix produced results most similar to the Miltenyi antibody method.
This finding reinforces the assertion of high purity with Norgen’s approach while being significantly more cost-effective than antibody purification methods.
Image Credit: Norgen Biotek Corp.
Loss of exosome integrity
Exosomes are fragile and susceptible to damage or alteration due to the harsh mechanical and chemical conditions often encountered in many commercial exosome isolation kits. Such degradation can negatively affect downstream applications, including next-generation sequencing (NGS).
Norgen addresses this concern through proprietary pH-based isolation technology, which modifies the isoelectric charges of the membrane-embedded proteins on the exosomes. This gentler approach minimizes damage or alteration to the exosomes, resulting in enhanced yield and purity.
Norgen facilitates efficient and reproducible exosome purification, focusing on the isolation of intact exosomes, exosomal RNA, or a combination of both from various sample types, including plasma, serum and urine.
The field of exosome research is rapidly evolving and holds significant potential for various applications in human health and disease. While this area shows great promise, numerous challenges remain to be addressed as the field continues to advance.
Acknowledgments
Produced from material originally authored by Jeremy Bannon from Norgen Biotek.
References and further reading
- Reale, A., et al. (2021). Human myeloma cell- and plasma-derived extracellular vesicles contribute to functional regulation of stromal cells. Proteomics, (online) 21(13-14), p.e2000119. https://doi.org/10.1002/pmic.202000119.
- Rolland, A. (2023). Why I use NORGEN. Cancer Treatment Options and Management (CTOAM).
About Norgen Biotek Corp.
Norgen Biotek: Advancing science with best-in-class, scientist-backed innovations
Norgen Biotek is a fully integrated biotechnology company that focuses on providing complete workflows for molecular biology sample preparation and analysis. With a diverse portfolio of over 600 products, the company delivers high-performance, user-friendly, and cost-effective solutions.
Scientifically driven, industry trusted
Norgen kits cover a broad range of applications from collection and preservation to isolation and purification. Our expert R&D team continuously develops cutting-edge technologies that set new industry standards for RNA, DNA, protein, and exosomal isolation, ensuring superior yield, purity, and integrity from even the most challenging sample types.
Unparalleled performance
At the heart of Norgen’s success is its patented Silicon Carbide (SiC) Technology. This proprietary resin exhibits uniform binding affinity for all RNA species, regardless of molecular weight or GC content. This ensures the full diversity of small and microRNA are captured while eliminating the need for phenol extraction. This innovative technology sets Norgen kits apart from others, positioning them as leaders in RNA purification.
Comprehensive solutions for any challenge
Norgen is committed to providing high-quality kits capable of processing a wide range of sample types, from ultra-low input samples such as liquid biopsies to highly impure samples like stool or soil. Our sample collection and preservation devices for stool and saliva simplify handling by rendering samples non-infectious by preventing microbial growth and inactivating viruses, while our blood and urine preservation solutions ensure the stability of highly vulnerable cell-free nucleic acids.
To meet varying research demands, we offer multiple isolation methods including, but not limited to high-throughput and automation-ready magnetic bead-based formats. Additionally, our multiple-analyte kits enable the simultaneous purification of RNA, DNA, and proteins, maximizing data extraction from a single sample.
Norgen offers an extensive variety of TaqMan qPCR kits designed for molecular diagnostic use, including lyophilized kits for easy shipping. Library preparation kits for both DNA and RNA samples are also available to support genomic applications. Norgen recently released their EXTRAClean technology, an innovative solution that minimizes background noise while providing high-purity RNA, significantly enhancing NGS performance.
Why choose Norgen?
- Scientist-Driven Innovation – Developed by leading experts in molecular biology
- Proven Quality & Reliability – ISO 9001 and ISO 13485 certified, indicating a commitment to selling high-quality products
- Global Presence – We ship to over 150 countries and have a network of 60+ distributors
- Award Winning – Norgen Biotek Corp. was honored with the 2021 Innovative Leaders Award, and recognized as the 2024 Rapid Star Award Winner in the PCR category.
- Innovative Products – Hold more than thirty issued and pending patents for products presenting solutions for all research & clinical applications
Driven by a mission to accelerate scientific discoveries, Norgen Biotek actively supports researchers by providing educational resources, technical workshops, and application notes. Their NorBlog serves as a hub for the latest scientific discoveries, protocol optimizations, and industry trends.
Explore Norgen Biotek’s innovative solutions today and take your research to the next level.
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