Enrichment of Native Lipoprotein Particles with microRNA and Subsequent Determination of Their Absolute/Relative microRNA Content and Their Cellular Transfer Rate

Lipoprotein particles are native transport vehicles. The enrichment of foreign substances facilitates their use as track carrier. Specific labeling of molecules or whole particles makes it feasible to measure the cellular uptake.

The two methods for enrichment are quick and easy to use, and can be adapted for a wide range of substances. Demonstrating the procedure will be Markus Axmann and Andreas Karner, two postdocs from my lab. To begin delipidation in a fume hood, mix one to two milliliters of prepared HDL solution containing five milligrams of HDL particles with 50 milliliters of pre-cooled three-to-two mixture of ethanol diethyl ether in a conical centrifugation tube.

Incubate for two hours at negative 20 degrees Celsius. Centrifuge at 2500 times g for 10 minutes at negative 10 degrees Celsius. Discard the supernatant, resuspend the pellet in 50 milliliters of pre-cooled ethanol diethyl ether mixture, and vortex briefly.

Incubate a second time for two hours at negative 20 degrees Celsius. Centrifuge again at 2500 times g for 10 minutes at negative 10 degrees Celsius. Then insert tubings supplying nitrogen gas flow to dry the pellet.

And resuspend it in 250 microliters of buffer A.Determine the protein concentration using the Bradford protein assay or another appropriate one. It is critical to remove any remnants of the ethanol diethyl ether mixture, as these solvents would inhibit the relipidation of apolipoproteins. Next, dilute to a final concentration of one milligrams of protein per 250 microliters of buffer A.Insert a tubing supplying inert gas into the solution part in the tube.

If required, store the solution overnight at four degrees Celsius under the inert gas atmosphere. To begin reconstitution, in a clean glass tube, mix 100 microliters of CO, 13.5 microliters of C, and 500 microliters of PC.Then rotate the glass tube while flushing nitrogen gas inside the tube to dry the mixture in order to yield a homogeneous surface layer. In a 0.5-milliliter reaction tube, prepare a fresh 30-millimolar spermine solution in buffer A.Then mix 100 microliters of 10-micromolar synthetic microRNA with 100 microliters of spermine solution in a two-milliliter reaction tube.

And incubate for 30 minutes at 30 degrees Celsius. Next, transfer the incubated 200-microliter solution into the glass tube to rehydrate the prepared PC-CO-C mastermix surface layer. And then add 50 microliters of a 30 milligrams per milliliter sodium deoxycholate solution.

Stir at four degrees Celsius for two hours. After that, add 250 microliters of the delipidated HDL solution into the glass tube. And stir at four degrees Celsius overnight.

To begin dialysis, first add 50 grams of adsorbent beads to 800 milliliters of double-distilled water. And use a magnetic stirrer to stir for one minute. Wait 15 minutes for the beads to settle, and decant the supernatant.

Repeat the procedure with pre-cooled PBS. Pre-wet dialysis cassettes in PBS. Use a syringe to add the previously-prepared mixture into the cassettes according to the manufacturer's instructions.

Add the PBS-treated adsorbent beads to three liters of PBS, and place the cassettes into the PBS to dialyze at four degrees Celsius. The beads keep the density gradient along the dialysis membrane constant. Change the buffer and the beads after one hour and two hours.

After 24 hours, with a syringe, extract the solution from the cassette into a 1.5-milliliter reaction tube to recover the reconstituted HDL particle solution. Determine the protein concentration using the Bradford assay. Supply inert gas to the reaction tube, seal it, and store the reconstituted HDL particle solution under inert gas atmosphere at four degrees Celsius.

First, prepare a fresh 30-millimolar spermine solution in RNAse-free water. Mix 100 microliters of 10 micromolar of synthetic microRNA with 100 microliters of spermine solution in a two-milliliter reaction tube. And incubate for 30 minutes at 30 degrees Celsius.

Then add 100 microliters of DMSO and 1.2 milliliters of 1X LDL buffer to the prepared microRNA spermine solution. Dilute previously-prepared LDL particle solution with PBS to a final concentration of approximately four milligrams per milliliter. Then draw 450 microliters of the diluted solution into a 1.5-milliliter reaction tube, and mix with 50 microliters of 10X LDL buffer.

Incubate it for 10 minutes on ice. After incubation, combine the 500-microliter LDL particle solution and the 1.5-milliliter microRNA-spermine-DMSO solution, and incubate it for two hours at 40 degrees Celsius. Perform dialysis similar as previously described, and store the labeled LDL particle solution under inert gas atmosphere at four degrees Celsius.

To begin, dilute the HDL or LDL particle solution in PBS between one-to-100 and one-to-1, 000. Cleave mica by pressing adhesive tape against the mica substrate, and pulling the tape off to remove the upper mica layers. Use a pipette to deposit two microliters on freshly-cleaved mica to incubate for five minutes.

Lipoprotein particles tend to form continuous bond membranes on surfaces. Consequently, it is important to adjust the particle concentration on mica surface to get individual ones. After incubation, rinse the sample with PBS.

Fill the high-speed AFM liquid cell with PBS, and mount the mica-carrying scanner to the high-speed AFM stage. In the control software, start the approach process of the cantilever to the mica surface. Use scan sizes less than one square micrometer, and keep the imaging forces as low as possible.

Image the sample in tapping mode. Load the data into the Gwyddion, and detect the particles via the mark grains by threshold function. Adjust the height threshold value to mask the individual particles.

Usually, a level around 50%is a good starting point. Remove polynomial background to flatten the image, and activate the exclude masked region option. Export the maximum height values of the detected particles using the distribution of various grain characteristics function, and repeat these steps for all recorded images.

In this experiment, reconstitution of HDL particles was performed through delipidation of HDL particles, followed by relipidation and dialysis. A yield of 50%of reconstituted HDL particles can be achieved. However, the same labeling with microRNA was not feasible for the labeling of LDL particles, due to the hydrophobicity of the apolipoprotein B100 protein.

Thus, DMSO was used for the penetration of the lipid monolayer of the LDL particles, with a yield close to 100%During quality control, the dilution is critical for analysis. The top image shows a too-high particle density. The bottom image is suitable for analysis.

Probability density functions of particle heights were calculated for comparison of size distributions of the native, labeled, and labeled control lipoprotein particles. The relative change before and after the labeling procedure is neglectable. When handling RNA oligonucleotides, work RNAse-free.

Use fresh disposable plastic consumables, and always wear gloves. Use only nuclease-free solutions. High-speed AFM is just one method to determine the general shape of lipoproteins.

An alternative method would be electron microscopy. Wear appropriate personal protection equipment, and work in a fume hood while handling diethyl ether.