Most health conditions can be better managed if detected early. Biomarkers available in accessible body fluids are the most promising ways for early detection of health conditions. We are reporting improvements in methods of investigating microRNA biomarkers released via small extracellular vesicles from human brain.
This method has allowed us to identify variation in small extracellular vesical cargoes between different genetic subtypes of disease, such as dementia, can identify cargo biomarker candidates that could be detected in peripheral fluids, and enhance early diagnosis of disease. So this protocol balances efficiency, accessibility, and product purity, making it easily reproducible across different laboratory environments. It doesn't require any specialized equipment and prepackaged columns provide consistency.
Therefore, any biomarker findings will be easily translated to a desired clinical outcome. With new disease modifying drugs being approved, diagnosis needs to take these diseases much earlier than currently possible to maximize patient benefit. Methods such as this will lead to the identification of new disease relevant microRNA biomarkers, which can result in better health management using specific treatment and care options.
We will use our existing capabilities to develop methods for identifying tissue and cell type-specific markers that can detect microRNA biomarkers released from a specific cell type within a heterogeneous mixture found in body fluids. To begin, thinly slice 250 milligrams of frozen brain tissue using a sterilized scalpel on a cold plate. Add two milliliters of 75 units per milliliter collagenase Type III in hibernate solution to the tissue.
Place each sample in a water bath at 37 degrees Celsius for 20 minutes. At the five-minute mark, invert the sample twice to mix. At the 10-minute mark, gently pipette the sample three times using a 10-milliliter plastic disposable stripette.
Then place each sample on ice, and add protease inhibitor cocktail, and phosphatase inhibitor to each sample. Centrifuge each brain tissue sample at 300G for 10 minutes at four degrees Celsius. Collect the supernatant.
And then centrifuge it at 2000G for 15 minutes at four degrees Celsius. Now collect the supernatant and filter it through a 0.22 micrometer filter. Then centrifuge the filtrate at 10, 000G for 48 minutes at four degrees Celsius.
After collecting the supernatant, add precipitation buffer to each sample in a two to one ratio. And incubate the sample overnight at four degrees Celsius. Then centrifuge each sample at 10, 000G for 96 minutes at four degrees Celsius.
And resuspend the pellet in 100 microliters of extracellular vesicle free PBS. Equilibrate the pre-prepared size exclusion chromatography or SEC column at room temperature for 15 minutes. After equilibration, remove the preservative buffer from the top of the column, and wash the column twice using 250 microliters of extracellular vesicle free PBS.
Then add the resuspended pellet to the column and centrifuge at 50G for 30 seconds. After discarding the flow through, add 180 microliters of EV free PBS, and centrifuge the column at 50G for one-minute to allow the brain derived small extracellular vesicles to elute. To lice small extracellular vesicles, dilute the isolated small extracellular vesicle sample in an equal ratio with lysis and extraction buffer.
Agitate the diluted sample at four degrees Celsius for 30 minutes. Then centrifuge the sample at 14, 000G for 15 minutes. Collect the protein supernatant and measure protein concentrations using a protein assay kit.
To perform nanoparticle tracking analysis, dilute each sample in PBS using a dilution factor of 1 to 1000. Inject the sample into the NTA instrument. And set the instrument to read the sample at a wavelength of 550 nanometers.
Use the particle quantity to measure the amount of protein per particle expressed in femto grams as per MISEV guidelines. Next, dilute the cell mask orange or CMO dye in PBS by a dilution factor of 1 to 1000. Then dilute the CMO dye with the small extracellular vesicle sample using a 1 to 10 dilution factor.
And incubate the mixture in the dark for 30 minutes at four degrees Celsius. Now, dilute the CMO dye small extracellular vesicle mixture further using PBS at a 1 to 1000 dilution factor. Using the NTA instrument, read the CMO dye of each small extracellular vesicle sample at a wavelength of 550 nanometers.
For fluorescent antibody tracking, dilute each tetraspan and fluorescent antibody in PBS using a 1 to 10 dilution factor. Then dilute each antibody dye with the small extracellular vesicle sample using a 1 to 10 dilution factor. And incubate the mixture in the dark for two hours at four degrees Celsius.
Next, dilute the second antibody mixture using PBS with a 1 to 1000 dilution factor. Read the fluorescent antibody signals of the small extracellular vesicle sample at a wavelength of 550 nanometers using the NTA instrument. Western blot analysis confirmed the presence of the positive markers CD9, CD63, CD81, Flotilla One, and TSG101 in brain-derived small extracellular vesicles, while Calnexin was absent indicating no cellular contamination.
Nanoparticle tracking analysis revealed that the isolated particles ranged between 50 to 200 nanometers in size with a peak concentration at around 100 nanometers. CMO staining data showed that 52.35%of the particles have a lipid bilayer, of which 34.66%contained CD9, 15.49%contained CD63, and 12.01%contained CD81.
