The overall goal of this procedure is to design a modified exosome precipitation method that offers a quick, scalable and effective alternative for the isolation of exosomes from urine. This is accomplished by first removing large dead cells and cell debris from the urine sample by centrifugation, and the snat containing the urinary exosomes is transferred to a fresh tube. In the second step, the pellet is dissolved in isolation solution containing DTT to remove TAM horse fall protein.
Then after further centrifugation, the TP free supernatant is incubated with exo quick TC reagent overnight to precipitate the exosomes the next day. The exo quick TC reagent treated samples are centrifuged one last time to obtain the exosome pellet. Ultimately, downstream processes such as Western blot and realtime QPCR can be performed on the exosomes to identify the biomarkers of interest.
The main advantage of this technique over existing methods like ultra centrification filtration and precipitation is arm method requires no ultra certification steps, is easy to perform relatively quick, while still providing a high, relatively pure exosome yield for subsequent molecular analysis Prior to urine collection. At 3.125 milliliters of freshly prepared protease inhibitor cocktail to an empty sterile receptacle, and then collect approximately 10 milliliters of first void urine in the receptacle. Transfer the sample into 15 milliliter conical tubes.
Please note, a 24 hour urine sample collected can also be used for exosome isolation by storing at four degrees Celsius. Spin the tubes for 10 minutes at 17, 000 GS and 37 degrees Celsius to remove or separate cellular debris from urinary exosomes. Next, taking care not to disturb the pellet, transfer the supernatant to a fresh tube and resuspend the pellet in 500 microliters of isolation solution.
Incubate the pellet with DTT at a final concentration of 200 milligrams per milliliter at 37 degrees Celsius for 10 minutes. Vortexing the sample every two minutes. Then when the pellet is completely dissolved, add 500 microliters of isolation solution to the sample and immediately spin down the resulting suspension.
Pool the supernatant with the previously collected supernatant and resuspend, the new pellet in 50 microliters of isolation solution. For SDS page analysis to isolate the exosomes, first incubate 11 milliliters of the pooled supernatant with 3.3 milliliters of exo quick TC reagent at four degrees Celsius for at least 12 hours. Following the incubation centrifuge the sample, then transfer the supernatant to a fresh tube for SDS page analysis and the pellet containing the urinary exosomes is used to extract D-N-A-R-N-A or protein, or stored at minus 80 degrees Celsius.
Use a NanoDrop spectrophotometer to determine the concentrations of DNA and RNA of each extraction by measuring the absorbence at 260 and 280 nanometers with two microliters of sample here. A modified reproduction of an SDS page analysis and biomarker detection using western blood analysis is shown in the lanes with unprocessed urine and the first urinary pellet protein bans corresponding to 90 to 100 kilodaltons were observed indicating the presence and the removal of the TAM horse fall protein respectively in the final supernatant and exosome pellet lanes. The TAM horse fall protein band was not observed indicating a removal of the TAM horse fall protein in the earlier steps and resulting in an increased exosome pellet yield.
In this experiment, biomarkers Alex and TSG 1 0 1 were detected even when low quantities of sample were used indicating a very pure exosome pellet for biomarker analysis and an absence of abundant soluble proteins, including the TAM horse fall protein After its development. This technique has paved the way for researchers in the field of chronic kidney disease to use urine isolated exosomes for the early detection and diagnosis of diabetic nephropathy.
