Discover microRNA and Protein Biomarkers in Patient Biofluids
Exosomes are 40 –100 nm membrane vesicles secreted by most cell types in vivo and in vitro. 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 exosome precipitation reagent makes microRNA and protein biomarker discoveries simple, reliable and quantitative. Enrich for circulating exosomal microRNAs with ExoQuick and accurately profile them using SBI’s QuantiMir qPCR arrays.
No time-consuming ultracentrifugation
Less expensive than costly Antibodies and beads
No complicated syringes required
Compatible with biofluid from any species
More effective than any other method
Isolate intact exosomes for functional studies
Use as little as 100 µl of serum or biofluid
How to use ExoQuick
Amounts of ExoQuick to add to various biofluids
Enrich for more exosomal proteins: Ascites and Urine
Isolate Exosomes and their Proteins from Ovarian Tumor Ascites Fluid
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 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.
Isolate more exosomal microRNAs
Isolate Exosome microRNAs from Ovarian Tumor Ascites Fluid
The 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.
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). The media from the cells was collected 48 h after plating and the exosomes from the media were precipitated using ExoQuick. 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.
