Exosome Isolation

Discover microRNA and Protein Biomarkers in Patient Biofluids and Culture Media

• Simple one-step precipitation
• No time-consuming ultracentrifugation
• No complicated syringes required
• Less expensive than costly antibodies and beads
• More effective than any other method
• Isolate intact exosomes for functional studies as well as microRNA and proteins
• Compatible with biofluid from any species
• Use as little as 250 µl of serum, plasma, or tumor ascites fluid, or 5 ml of media, spinal fluid, or urine

Exosomes are 40 –100 nm membrane vesicles secreted by most cell types in vivo and in vitro. Their cargo reflects the origin and pysiological stet of the source cells.
Exosomes are found in blood, urine, amniotic fluid, malignant ascite fluids and contain distinct subsets of microRNAs depending upon the tumor from which they are secreted. SBI´s ExoQuick and ExoQuick-TC polymer-based exosome precipitation reagents make microRNA and protein biomarker discoveries simple, reliable and quantitative.

Circulating exosomes isolated by ExoQuiock and ExoQuick-TC yield exosomal RNA and protein with greater purity and quantity than chromatography, ultracentrifugation or DynaBeads. This enhances the senitivity and accuracy of downstream applications, such as qRT-PCR Profiling of miRNA and mass spectrometric or electrophoretic analyses of exosomal proteins.
Enrich for circulating exosomal microRNAs with ExoQuick or ExoQuick-TC (depending on the sample type) and accurately profile them using QuantiMir qPCR arrays.




Exosome Precipitation using ExoQuick


Isolation of Exosome microRNAs from Ovarian Tumor Ascites Fluid

Exosomes were isolated with different methods as indicated.
Exosomal RNA was purified and RNA quality and yield was accessed using a GeneQuant II. Small RNAs were analyzed with the Agilent 2100 Bioanalyzer Lab-on-a-Chip instrument system (Agilent Technologies, Santa Clara, CA), using the Agilent Small RNA chip and reagent kit. Approximately 100ng of isolated total RNA in 1μl was applied to each run. The manufacturer’s recommended protocol was strictly followed to obtain Bioanalyzer profiles for the size range 6 to 150 nucleotides (nt). The profiles were calibrated for size (nt) using the small RNA ladder supplied with the kit, containing markers of 20, 40, 60, 80, and 150 nt in size, as reference. The instrument software quantitated the peak area between 0 and 150 nt as small RNA region, the area within 10 to 40 nt as microRNA region, and provides percentages of miRNA detected for each sample.

Isolation of Exosome Proteins from Ovarian Tumor Ascites Fluid


Enrichment of Placental Alkaline Phosphatase
Exosomes were isolated with different methods as indicated.
The quantity of protein was determined by the Bradford microassay method (Bio-Rad Laboratories), using BSA as a standard. Proteins from each exosome isolate were standardized to the original sample volume and equal volumes were applied per lane of a 12.5% SDS-PAGE gel. Western immunoblotting was performed to analyze the presence of the specific marker protein, placental alkaline phosphatase (PLAP). The SDS-PAGE gel was transferred to a nitrocellulose membrane, the membrane blocked for 1 hour at room temperature with non-fat dried milk, and probed overnight at 4°C with primary antibody. The bound immune complexes were visualized by enhanced chemiluminescence (ECL, Amersham Life Sciences) and quantitated by densitometry (Un-Scan-it Software, Silk Scientific Corp.).

Human Urine Exosome Protein Biomarker Analysis

Human urine samples (1 ml) were treated with 1 ml ExoQuick. Exosome pellets recovered were resuspended in 35µl PBS and the supernatants used as controls. Equal volumes (10µl) of urine supernatant (Sup) and exosome pellets (EXO) were separated on 4–15% gradient PAGE gels (Bio-Rad). Standard Western blot procedures with antibodies to Annexin V and Aquaporin-2 (both from Abcam, Inc.) were used to detect urine exosomal protein biomarkers.


ExoQuick isolated exosomes are functional


ExoQuick exosomes can be transfered between cells.
SBI created a stable 293TN cell line overexpressing the Cyto-Tracer™ pCT-CD63-GFP fusion protein (catalog# CYTO120-PA-1-SBI). The media from the cells was collected 48 h after plating and the exosomes from the media were precipitated using ExoQuick-TC. The exosome pellet recovered was resuspended in 30ul PBS and 10ul was added to newly plated HT1080 cells. HT1080 cells were visualized 72 h after the addition of the CD63-GFP labeled exosomes and then re-plated. Following another 24h, the cells were again visualized for GFP fluorescence and imaged. The exosomes appear to dock with the cells within 72 h and some are found to be internalized after 96 h.

Product Citations
Taylor, D. D. et al. (2011) Exosome Isolation for Proteomic Analyses and RNA Profiling. Serum/Plasma Proteomics, Methods in Molecular Biology, Vol. 728, Part 4, 235-246; see link below

Karolina et al. MicroRNA 144 Impairs Insulin Signaling by Inhibiting the Expression of Insulin Receptor Substrate 1 in Type 2 Diabetes Mellitus. PLoS ONE 6(8): e22839. doi:10.1371/journal.pone.0022839

Tae Hoon Lee et al. Review: Microvesicles as mediators of intercellular communication in cancer—the emerging science of cellular ´debris´. Seminars in Immunopathology DOI: 10.1007/s00281-011-0250-3

References
Mitchell, PS et al. (2008) Circulating microRNAs as stable blood-based markers for cancer detection. Proc Natl Acad Sci U S A. 105(30):10513-8.
Laterza, OF et al. (2009) Plasma MicroRNAs as sensitive and specific biomarkers of tissue injury.Clin Chem. 55(11):1977-83.
Valadi, H et al. (2007) Exosome-mediated transfer of mRNAs and microRNAs is a novel mechanism of genetic exchange between cells. Nat Cell Biol. 9(6):654-9.
Pegtel, DM et al. (2010) Functional delivery of viral miRNAs via exosomes. Proc Natl Acad Sci USA 107(14):6328-33.
Mathivanan, S and Simpson, RJ (2009) ExoCarta: A compendium of exosomal proteins and RNA. Proteomics. 21: 4997-5000.
Théry, C et al. (2009) Membrane vesicles as conveyors of immune responses. Nat Rev Immunol. 8:581-93.
Michael, A et al. (2010) Exosomes from human saliva as a source of microRNA biomarkers. Oral Dis 16(1):34-8.
Luo, SS et al. (2009) Human villous trophoblasts express and secrete placenta-specific microRNAs into maternal circulation via exosomes. Biol Reprod 81(4):717-29. Taylor, DD and Gercel-Taylor, C (2008) MicroRNA signatures of tumor-derived exosomes as diagnostic biomarkers of ovarian cancer. Gynecol Oncol 110(1):13-21.
Simpson, RJ et al. (2009) Exosomes: proteomic insights and diagnostic potential. Expert Rev Proteomics. 6(3):267-83. Review.

NanoSight Data showing the exosome isolation with ExoQuick
Data showing biological activity of the purified exosomes
Taylor et al.: Abstract about Exosome Isolation
Combination Kit: ExoQuick plus exoRNA Purification Columns
Exosomal microRNA Profiling from Serum
QuantiMir qPCR Arrays
Exosome Labeling using Cyto-Tracer GFP-Fusion Vector
Exocarta online database of exosome proteins and RNAs

Product Brochure ExoQuick
Product Brochure ExoQuick-TC
User Manual ExoQuick
User Manual ExoQuick-TC

Description	Cat#	Size	Price
ExoQuick Exosome Precipitation Solution for Serum, Plasma and Tumor Ascites Fluid (5 ml)	EXOQ5A-1-SBI	75 rxns (250 ul sample volume)	325 €	DETAILS
ExoQuick Exosome Precipitation Solution for Serum, Plasma and Tumor Ascites Fluid (20 ml)	EXOQ20A-1-SBI	300 rxns (250 ul sample volume)	941 €	DETAILS
ExoQuick-TC Exosome Precipitation Solution for Culture Media, Spinal Fluid and Urine (10 ml)	EXOTC10A-1-SBI	10 rxns (5 ml sample volume)	324 €	DETAILS
ExoQuick-TC Exosome Precipitation Solution for Culture Media, Spinal Fluid and Urine (50 ml)	EXOTC50A-1-SBI	50 rxns (5 ml sample volume)	974 €	DETAILS