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 (see also special kit), or tumor ascites fluid, or 5 ml of media, spinal fluid, or urine
• Exosome depleted FBS available to avoid bovine derived exosomes in your experiments
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 ExoQuick 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.
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
Zhou Q et al. Immune-related MicroRNAs are Abundant in Breast Milk Exosomes. Int J Biol Sci 2012; 8:118-123
Yaron Goren et al. Serum levels of microRNAs in patients with heart failure. Eur J Heart Fail. published 25 November 2011, 10.1093/eurjhf/hfr155
Haney MJ et al. Blood-borne macrophage-neural cell interactions hitchhike endosome networks for cell-based nanozyme brain delivery. Nanomedicine (Lond). 2012 Jan 11. [Epub ahead of print]
Da Silveira JC et al. Cell-Secreted Vesicles in Equine Ovarian Follicular Fluid Contain miRNAs and Proteins: A Possible New Form of Cell Communication Within the Ovarian Follicle. Biol Reprod. 2011 Nov 23. [Epub ahead of print]
Hartman ZC et al. Increasing vaccine potency through exosome antigen targeting. Vaccine. 2011 Oct 12. [Epub ahead of print]
Karolina DS 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
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.
New! Exo-FBS™ Exosome-depleted FBS
• Exosome-sized vesicles removed
• FBS that has been stripped of CD63 positive cow exosomes
• No detectable bovine microRNAs
• Same cellular growth rates supported as standard FBS
• Use the same as standard FBS (add 10% in DMEM or RPMI)
Standard growth medium for most cells in culture require fetal bovine serum (FBS) as a growth supplement to DMEM. FBS is derived from bovine (cow) serum and contains a high abundance of cow exosome vesicles. These exosomes can interfere or cause significant background issues when studying the exosomes secreted from your cells of interest in standard culture conditions.
Our partner SBI has developed an exosome-depleted FBS growth supplement called Exo-FBS that has been stripped of bovine exosomes. Exo-FBS supports equivalent growth of many types of cells in culture, is devoid of cow CD63 positive exosomes and does not have any measurable bovine microRNAs. Perform your studies of cellular secreted exosomes in culture without the worry of contaminating cow exosomes in your experiments. No ultracentrifugation required. Available as standard FBS supplement or heat inactivated FBS media supplement (treated at 65°C for 15 minutes before bovine exosome removal).
For more information see individual Exo-FBS products and product brochure (link below).
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
Combination Kit: ExoQuick-TC & 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
|ExoQuick Exosome Precipitation Solution for Serum, Plasma and Tumor Ascites Fluid||EXOQ5A-1-SBI||75 rxns (250 ul sample volume)||
|ExoQuick Exosome Precipitation Solution for Serum, Plasma and Tumor Ascites Fluid (20 ml)||EXOQ20A-1-SBI||300 rxns (250 ul sample volume)||
|ExoQuick Plasma prep and Exosome precipitation kit (5 ml ExoQuick plus 100x Thromboplastin D)||EXOQ5TD-1-SBI||75 rxns (250 ul sample volume)||
|ExoQuick-TC Exosome Precipitation Solution for Culture Media, Spinal Fluid and Urine (10 ml)||EXOTC10A-1-SBI||10 rxns (5 ml sample volume)||
|ExoQuick-TC Exosome Precipitation Solution for Culture Media, Spinal Fluid and Urine (50 ml)||EXOTC50A-1-SBI||50 rxns (5 ml sample volume)||
|Exosome-depleted FBS Media Supplement||EXO-FBS-50A-1-SBI||50 ml||265 €||DETAILS|
|Exosome-depleted FBS Media Supplement||EXO-FBS-250A-1-SBI||250 ml||765 €||DETAILS|
|Exosome-depleted FBS Media Supplement Heat Inactivated||EXO-FBSHI-50A-1-SBI||50 ml||265 €||DETAILS|
|Exosome-depleted FBS Media Supplement Heat Inactivated||EXO-FBSHI-250A-1-SBI||250 ml||765 €||DETAILS|
|Thromboplastin D Plasma prep for Exosome precipitation (100x Thromboplastin D )||TDEXO-1-SBI||100 reactions||485 €||DETAILS|