In the rapidly evolving field of molecular diagnostics and precision medicine, exosomes and their cargo have emerged as critical disease biomarkers. These tiny extracellular vesicles, found in bodily fluids like serum and urine, offer valuable insights into gene expression, disease progression, and therapeutic response. However, isolating them with precision remains a significant challenge.

Enter Norgen Biotek’s Silicon Carbide (SiC) Technology. A revolutionary platform that promises high-purity, intact exosomes with no bias, contamination, or time-consuming processes. This blog explores how Norgen's patented method outperforms other commercial as well as traditional isolation techniques and is validated through peer-reviewed scientific research.

Understanding Exosomes and Exosomal RNA

What are Exosomes and their biological function?

Exosomes are a type of extracellular vesicles (EVs) in the size range 40 nm to 100 nm. They are lipid-membrane bound and contain cellular materials such as proteins, lipids, mRNAs, miRNAs, etc., as their cargo.¹ EVs, especially exosomes, play a crucial role in cellular communication and several biological processes. For example, exosomes are associated with immune response, viral pathogenicity, pregnancy, cardiovascular diseases, central nervous system-related diseases, and cancer progression. The receiver cell's response can be altered by exosomes when they deliver molecules such as proteins, nucleic acids, or metabolites. These altered responses can lead to disease promotion or disease restriction. ²

What are Exosomal Proteins?

Exosomal proteins are one of the essential cargoes carried by exosomes. They can be found embedded on the surface of the exosome membrane or encapsulated within the membrane. These proteins serve a wide range of biological roles: structural scaffolding, cargo trafficking, signal transduction, immune modulation, and even direct enzymatic activity. For example, heat shock proteins (HSP70, HSP90) and cytoskeletal elements (actin, tubulin) are frequently found in exosomes and contribute to vesicle stability, folding of proteins, and transport. ¹²

What is Exosomal RNA?

Exosomes carry various types of RNA, including mRNA, miRNA, lncRNA, and circRNA. Exosomal RNAs act as important regulators for cellular processes. They can be found throughout the body and are highly stable due to the membrane of the exosome keeping them safe from RNases. Exosomal RNAs are critical biomarkers for cancers and an excellent tool for evaluating drug resistance and diagnosing other diseases, such as those related to the central nervous system and urinary system. ³

Given the diagnostic and regulatory importance of exosomal protein and RNA, the ability to reliably isolate these vesicles from biological fluids becomes critically important. Accurate exosome isolation is the first step toward downstream protein and RNA analysis, biomarker discovery, and clinical application.

Exosome Isolation Methods

Ultracentrifugation

Ultracentrifugation has long been one of the most commonly used methods for isolating exosomes from biological samples. This technique relies on high-speed centrifugal forces to separate exosomes based on their size and density. It involves multiple rounds of differential centrifugation, gradually removing larger particles before finally pelleting the exosomes at speeds exceeding 100,000 × g. ⁷

While ultracentrifugation was once considered a standard approach, recent advancements in exosome isolation have exposed its inefficiencies, making it increasingly obsolete. Despite its historical relevance, its vulnerability to impurities, long processing times, and high equipment costs make it a less favorable choice compared to modern alternatives. ⁸

Polymer Precipitation

Polymer precipitation is a widely used method for exosome isolation due to its simplicity and scalability. This technique relies on hydrophilic, water-excluding polymers such as polyethylene glycol (PEG), which alter the solubility of exosomes and lead to their precipitation. The method is commonly used in commercial exosome isolation kits and is applied to a variety of biological fluids, including blood, urine, and cerebrospinal fluid. ⁹

Despite its accessibility, polymer precipitation presents several drawbacks that limit its effectiveness in exosome isolation. The technique frequently results in contamination from non-exosomal components, interferes with downstream analyses, and often requires additional purification steps. ⁹ These limitations make polymer precipitation a suboptimal choice when compared to more advanced isolation techniques, which can provide higher purity and efficiency.

Size-Exclusion Chromatography (SEC)

Size-Exclusion Chromatography (SEC) is a technique that separates particles based on their size by passing a biological sample through a stationary phase packed with porous resin particles. Larger particles, such as exosomes, elute earlier, while smaller contaminants are retained longer within the pores. This gentle process helps maintain the integrity of exosomes during isolation. ⁹

While Size-Exclusion Chromatography offers a gentle method for exosome isolation, its limitations, including co-isolation of contaminants, limited sample capacity, and potential sample dilution, reduce its appeal compared to more efficient modern techniques.

Silicon Carbide (SiC) Technology

Norgen’s Silicon Carbide (SiC) technology is a proprietary exosome purification method designed for a fast, simple isolation protocol with minimal contamination. Unlike traditional approaches, SiC selectively binds exosomes at the exosome membrane proteins’ isoelectric points, ensuring efficient separation without harsh chemical reagents or high-speed centrifugation. ¹⁰ This method delivers high-purity exosomes in under 30 minutes, making it an ideal solution for research.

Advantages of Silicon Carbide

1. High Purity and Contaminant-Free Isolation
   • Selective pH-based binding ensures efficient removal of protein aggregates, nucleic acids, and non-vesicular contaminants, producing a cleaner exosome fraction than polymer precipitation and ultracentrifugation.
   • Unlike polymer-based precipitation kits, which often leave behind chemical residues (e.g., PEG), SiC eliminates the need for harsh additives, ensuring high sample purity.
   • According to a study comparing exosome isolation methods, SiC-based purification significantly reduces contamination from ribosomes and macromolecular complexes, yielding higher RNA purity than ultracentrifugation or polymer-based kits. ¹⁰
   • Another study used Norgen’s Plasma/Serum Exosome Purification Kit and found “the presence of exosomal markers CD63 and CD81 and negative for calnexin and any endoplasmic reticulum contamination.” (Singh et al., 2020) ¹³
2. Rapid Scalable Protocol
   • Exosome isolation is completed in under 30 minutes, significantly reducing processing time compared to ultracentrifugation (4–6 hours).
   • The protocol is easily scalable, allowing researchers to process different sample volumes without altering efficiency or requiring expensive equipment.
3. Preserves Exosome Integrity for Downstream Analysis
   • The gentle pH-based capture and release mechanism prevents mechanical stress fragmentation, maintaining exosomal structure and biological activity.
   • TEM (Transmission Electron Microscopy) analysis confirms that SiC-isolated exosomes retain their morphology, making them ideal for RNA sequencing, mass spectrometry, and proteomics research.

Compatibility Across Biofluids

Exosomes exist in abundance in different biofluids such as plasma/serum, urine, saliva, and more. One of the defining strengths of Norgen Biotek’s Silicon Carbide (SiC) exosome isolation platform is its broad compatibility with diverse biological sample types.

Exosomes from Plasma/Serum

Plasma and serum remain gold-standard biofluids for liquid biopsy applications due to their high systemic relevance and accessibility in clinical settings. Exosomes isolated from these fluids can be profiled for small RNAs, mRNA transcripts, and surface proteins, supporting diagnostics and longitudinal monitoring.

Urinary Exosomes and Exosomal mRNA

The collection process for urine is convenient, non-invasive, and ideal for mass screening and early detection of conditions like cancer. Additionally, exosomal mRNA is an excellent biomarker due to the lipid bilayer of exosomes, which ensures genetic information inside is protected from degradation by nucleases.

A recent study evaluated the potential of urinary exosomal mRNA as a non-invasive biomarker for early prostate cancer detection. Using Norgen’s Urine Exosome RNA Isolation Kit and next-generation sequencing, researchers identified ten candidate mRNAs from urinary exosomes of prostate cancer patients. Two of which, WWP1 and RAB5B, emerged as the most diagnostically valuable. A logistic regression model built on these two genes achieved an AUC of 0.923, with 81.4% sensitivity and 89.1% specificity, outperforming traditional Prostate-Specific Antigen (PSA) testing. The authors concluded that urinary exosomal mRNA profiling represents a promising, non-invasive strategy for improving the early diagnosis of prostate cancer, especially when collected after digital rectal examination (DRE). ⁶

Scientific Validation Through Peer-Reviewed Article

Comparative Studies Place Norgen on Top

In a recent comparative study conducted by Reseco et al. in 2024, Norgen’s Saliva Exosome Purification Kit demonstrated superior performance compared to other commercially available kits. This kit achieved recovery rates of up to 97% in younger adults and 86% in older adults after magnetic bead immunocapture. Notably, this kit is effective in capturing smaller EVs (30–50 nm) with critical biomarkers like CD63 and CD81. With a rapid turnaround time of 0.8 hours, minimal hands-on effort (25 mins), and significant cost-efficiency, Norgen offers a reliable and practical solution for consistent and effective EV research. ⁴

Moreover, Reale et al. conducted a proteomic study of plasma-derived small extracellular vesicles (psEVs) to highlight the critical role of pre-analytical factors in psEV isolation. Among the tested commercial kits, the Norgen’s Plasma/Serum Exosome Purification Kit stood out for its ability to selectively enrich the exosomal marker TSG101, as confirmed by Western Blotting, despite the presence of abundant plasma proteins like human serum albumin (HSA). Transmission Electron Microscopy (TEM) confirmed the presence of characteristic cup-shaped vesicles (50–180 nm), while nanoparticle tracking analysis revealed a consistent size distribution across all tested isolation methods. Importantly, the purified exosomes were also successfully subjected to mass spectrometry-based proteomic analysis, demonstrating that the Norgen kit preserves both protein content and vesicle integrity. Based on these strengths, the authors selected the Norgen method for subsequent studies evaluating how pre-analytical variables play a critical role in psEV isolation.¹¹

Want to see how Norgen’s Plasma/Serum Exosome Purification kit works in real time? This short video demonstrates the user-friendly workflow, highlighting how easy it is to isolate intact extracellular vesicles suitable for downstream analysis.

Norgen's Kit Enables Functional Exosome Isolation

In another recent publication in the Journal of Cellular and Molecular Medicine, Liang et al. (2024) used Norgen Biotek’s Cell Culture Media Exosome Purification Kit to isolate tumor-derived exosomes from estrogen-treated ovarian cancer cell lines. They found that human ovarian cancer cell lines SKOV3 and ES2 increased exosome secretion with estrogen exposure. This was confirmed by high levels of canonical exosomal markers (CD63, CD9, and CD81). Their presence indicates that the exosomes were not only successfully isolated but also biologically intact, with preserved surface protein composition. ⁵

To verify the quality of the isolated exosomes, the researchers confirmed both their morphology and their molecular signature. Specifically, they used TEM and Western Blotting to validate that the vesicles were structurally intact and displayed hallmark exosomal surface markers. The authors demonstrated that estrogen treatment increased the expression of these markers, indicating that Norgen’s kit was sensitive enough to detect physiological changes in exosome production. ⁵

MCF-10A cells fluoresce green after incubation with mda-mb-231 exosomes.

According to a study conducted by Tokgün et al. in 2023, exosomes purified with Norgen's system are not only structurally intact but also biologically active. The researchers transfected the MDA-MB-231 cell line with a GFP-fused CD63. Exosomes were harvested from cells that expressed CD63-GFP and incubated with MCF-10A cells. They then used fluorescence microscopy to analyze the cells. According to the results, the MCF-10A cells fluoresced green, indicating successful uptake of the exosomes and the retention of biological activity. ¹⁴

Conclusion

Taking exosome isolation to the next step requires overcoming key technical challenges like purity, yield, and consistency. Norgen Biotek’s innovative Silicon Carbide (SiC) technology addresses these challenges head-on, offering unparalleled purity, high recovery rates, and rapid turnaround. Scientifically validated through rigorous peer-reviewed research, this technology represents a significant advancement for exosomes, exosomal proteins, and RNA isolation, paving the way for accurate and reliable molecular diagnostics, biomarker discovery, and precision medicine.

FAQs

Why are exosomes valuable for disease research?

They carry cellular information, making them ideal for monitoring disease progression.

Can I use this technology with saliva or urine samples?

Yes. Norgen’s kits are compatible with a variety of biofluids, including urine, saliva, plasma, and more.

Do I need phenol or carrier RNA during extraction?

No. Norgen’s platform is free from these reagents.

How fast is the isolation process?

Complete exosome isolation is achievable in under 30 minutes.

Is this system suitable for RNA sequencing?

Yes. It produces high-purity RNA ready for downstream applications like NGS.