The overall goal of this method is to develop a simplified approach to rapidly isolate purified high density lipoproteins from human blood plasma for micro RNA analysis. This method can help answer key questions in hepatology and cardiology, such as understanding the molecular mechanisms and protective functions of high density lipoproteins. Some main advantages of this technique is that purified HDL micro RNA come pre-isolated from less volume of samples within a short period of time.
In this protocol, plasma is obtained from fasting peripheral venous blood samples by multiple centrifugation steps as described in the protocol text. Transfer the plasma to a new tube. And measure the density of the plasma using a densitometer at room temperature, per the manufacturer's instructions.
To remove the circulating exosomes that represent a source of micro RNA contamination, add 252 microliters of exosome precipitation solution to one milliliter of plasma. And incubate for 30 minutes at four degrees Celsius. Then centrifuge the mixture for 30 minutes to pellet out the exosomes.
Transfer one milliliter of the resulting supernatant to a polycarbonate thick walled ultracentrifuge tube for further processing, as demonstrated in the next video segment. Isolation of high density lipoproteins, or HDL, is accomplished by a three step density gradient ultracentrifugation protocol, that involves a sequential isolation of very low density lipoproteins, or VLDL, low density lipoproteins, or LDL, and HDL. The three different density solutions, A, B, and C, must be prepared fresh, following the procedure described in the protocol text.
In a 6.5 milliliter polycarbonate thick walled ultracentrifuge tube, mix one milliliter of plasma and 200 microliters of nuclease-free Fat Red 7B. Then, carefully layer five milliliters of solution A on top of the mixture. If needed, add additional Fat Red 7B on top of solution A to balance the weight of each tube.
Load the tubes into a pre-chilled rotor. Place the rotor into a floor centrifuge and centrifuge for two hours. At the end of the run, two layers should be visible.
Remove 1.5 milliliters of the top layer, representing the VLDL fraction. And store it four degrees Celsius. Use a pipette to transfer four milliliters from the bottom of the tube to a new 50 milliliter tube.
This contains the LDL and HDL fraction. The next step is the isolation of LDL. Mix two milliliters of solution B and 100 microliters of nuclease-free Fat Red 7B into the tube containing the LDL and HDL fraction.
Put on ice for five to ten minutes. Transfer 6.5 milliliters of the mixed sample into a polycarbonate thick walled ultracentrifuge tube. Load the tube into a pre-chilled rotor and centrifuge for three hours.
When the centrifugation is complete, remove 1.5 milliliters of the top layer, representing the LDL fraction and keep at four degrees Celsius. Transfer four milliliters from the bottom of the tube, containing the HDL fraction to a 50 milliliter sterile tube. The third step is the isolation of HDL.
Mix two milliliters of solution C, 100 microliters of nuclease-free Fat Red 7B, and 15 microliters of 98%beta mercaptoethanol into the tube, containing the HDL fraction. Check the density of the mixture. Load the tube into a pre-chilled rotor and centrifuge for three hours.
After that, remove two milliliters of the top layer, representing the HDL fraction, and keep at four degrees Celsius. Excessive salt must be removed from the lipoprotein fractions to avoid interference with subsequent agarose gel electrophoresis and PCR. In this video, desalting and concentration will be demonstrated for only the HDL fraction.
Add 13 milliliters of cold PBS to the HDL fraction and transfer to a centrifugal filter device with the molecular weight cut-off of 10K. Centrifuge in a swinging bucket rotor, at 4000 times g, and four degrees Celsius, for 30 minutes. Desalt the HDL fraction, a second time, using 13 milliliters of cold PBS, and centrifuging as before.
After centrifugation, remove the lipoprotein containing solutes, and keep at four degrees Celsius. To assess the quality and purity of the isolated lipoproteins, agarose gel electrophoresis was performed with human lipoprotein standards, included as a size reference. Representative results from an isolation, without beta mercaptoethanol, showed that the HDL fraction is devoid of any contamination of VLDL and exhibits typical alpha mobility.
However, the HDL fraction also shows traces of beta mobility due to contamination with lipoproteins. The addition of beta mercaptoethanol to solution C, during the last centrifugation step, effectively removes all contaminating lipoproteins from the HDL fraction. To demonstrate the feasibility of quantifying micro RNAs, carried in human HDL, purified with this method, two different micro RNAs were amplified by real-time quantitative PCR.
In these amplification plots, the horizontal lines represent the detection threshold. Representative real-time PCR data from isolated HDL, obtained from six samples, showed the consistent detection of both micro RNAs. Lastly, melting curve analyses clearly show a distinct single peak for both micro RNAs, consistent with specific amplification in the preceding PCR.
Once mastered, this technique can be done in nine hours, together with the desalting procedures, if it is done properly. While attempting this procedure, it is important to work at four to eight degrees centigrade, at all times. And to adjust the density of each lipoprotein fraction before ultracentrifugation.
Following this procedure, other methods, such as electron microscopy, can be performed in order to answer additional questions, like the purity of HDL. After its development, this technique will allow researchers, in the field of metabolic syndrome, to explore HDL-associated micro RNAs as bio markers, in humans with suspected fatty liver disease. It may also be used to better understand the function of HDL and the molecular mechanisms by which it alters cell biology.
